Chuyển gen vào ngô

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Biến nạp gen ở ngô (Zea mays L.) bằng nuôi cấy mô hạt từ dòng tự phối R23 đã thực hienẹ bằng bắn gen với một cấu truc mang CBF3 dưới sự điều khiển hoạt hóa của rd29A và marker chọn lọc hygromycin phosphotransferase. CBF3 đã biểu hiên tăng cưonưg tính chịu lạnh, chịu mặn và chịu hạn ở cây Arabidopsis, thuốc lá (Nicotiana tabacum L.), và lúa mỳ (Triticum aestivum L.).

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Published online 7 November 2007 Published in Crop Sci 47:2390-2402 (2007) © 2007 Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY Genetic Engineering of Maize with the Arabidopsis DREB1A/CBF3 Gene Using Split-Seed Explants Kỹ nghệ di truyền của ngô với gen DREB1A/CBF3 Arabidopsis sử dụng nuôi cây mô hạt Diaa Al-Abedb, Parani Madasamya, Reddy Tallaa, Stephen Goldmana and Sairam Rudrabhatlac,* Biến nạp gen ở ngô (Zea mays L.) bằng nuôi cấy mô hạt từ dòng tự phối R23 đã thực hienẹ bằng bắn gen với một cấu truc mang CBF3 dưới sự điều khiển hoạt hóa của rd29A và marker chọn lọc hygromycin phosphotransferase. CBF3 đã biểu hiên tăng cưonưg tính chịu lạnh, chịu mặn và chịu hạn ở cây Arabidopsis, thuốc lá (Nicotiana tabacum L.), và lúa mỳ (Triticum aestivum L.). Gen CBF3 đã dò tìm trng 18 dòng bằng PCR và nhân lên ổn định được 3 dòng khăng định bằng phân tích Southern blot. Phiên mã ngược PCR tìm biểu hiện của CBF3 trong các dòng chuyển gen dưới điều kiện không bất thuận mặc dù sử dụng đoạn hoạt hóa rd29A. Tổ hợp biểu hiện đã liên kết với chậm sinh trưởng và bất thụ ở hầu hết các dòng chuyển gen. Sự truyền của gen trong mo hinh Mendel đến thế hệ T1 và T2 đã được xác định trong 01 dòng bằng phân tích Southern blot. Các cây trong dòng này biểu hiên chịu bất thuận của gen CBF3 và có thể dò tmf được trong điều kienẹ không bất thuận , chống chịu lạnh, hạn và mặn so với các cây hoang dại. Kết quả này chứg minh CBF3 có tiềm năng nâng cao khả năng chống chịu bất thuanạ phi sinh học của ngô. Từ viết tắt: DRE/CRT, dehydration responsive element/c-repeat element • GUS, ß- glucuronidase gene • MS, Murashige and Skoog • MSI, multiple shoots induction • PCR, polymerase chain reaction • RT-PCR, reverse transcription polymerase chain reaction INTRODUCTION MAIZE IS ONE of the most important commercial crops in the world and is valued for its food, fiber, oil, and other byproducts. Because grain demand could outpace supply, particularly during dry or cold seasons, producing maize cultivars that are drought, cold, and salt tolerant will help ensure grain capacity and price stability. Furthermore, it may allow an expansion in maize acreage, providing farmers with more planting options. Classical plant breeding protocols have been successfully used to develop lines adapted to survive under or at least mitigate against abiotic and biotic challenges. This technology has a number of disadvantages, most particularly the time needed to select and expand the foundation seed parent(s) (Holmberg and Bülow, 1998). To further enhance productivity by
bringing cultivars to market, the emerging technologies of gene transfer are being used as a complement to traditional breeding practices. Recently, it was reported that the Arabidopsis CBF transcriptional factors family interact with the dehydration responsive element/c-repeat element (DRE/CRT). The DRE/CRT is a cis-acting promoter element that regulates gene expression in response to drought, salt, and cold stress in Arabidopsis (Yamaguchi-Shinozaki and Shinozaki, 1994). Furthermore, expression of this gene enhances drought, salt, and cold tolerance in a number of plant species (Yamaguchi-Shinozaki and Shinozaki, 1994; Stockinger et al., 1997; Kasuga et al., 1999; Gilmour et al., 2000; Pellegrineschi et al., 2004). The main function of the CBF regulon is to protect the plant cells from freezing and water stress (Thomashow, 2001). The CBF regulatory proteins work as switches that activate multiple genes of the cold acclimation and water stress responses (Thomashow, 2001). A number of genes were identified in Arabidopsis to contain the DRE/CRT element, including: KIN1, COR6.6/KIN2, COR15a, COR47/RD17, COR78/RD29a, and ERD10 (Kasuga et al., 1999). These target genes encode for proteins that work on stabilizing cell membranes when subjected to different stress types. In addition, overexpression of CBF3 in Arabidopsis led to increased proline and total sugar levels and, in turn, enhanced freezing tolerance (Gilmour et al., 2000). The DRE in the rd29A promoter also functions in gene expression in response to stress in tobacco plants (Yamaguchi-Shinozaki and Shinozaki, 1994), which suggests that the rd29A promoter regulates the expression of DREB in tobacco as in Arabidopsis. These results indicate that the stress-inducible rd29A promoter is quite useful to overexpress CBF3 for improving drought, salt, and freezing stress tolerance not only in transgenic Arabidopsis but also in other kinds of transgenic plants such as tobacco (Kasuga et al., 2004). The application of transgenic technologies to maximum effect, however, requires overcoming certain limitations. These include restrictions with respect to genotype (Tomes and Smith, 1985), speed (Vasil et al., 1984; Bhaskaran and Smith, 1990), number of regenerants (Huang and Wei, 2004), and somaclonal variation and albinism (Armstrong and Phillips, 1988). Given these limitations, it becomes mandatory to link a DNA transfer technology to the chosen tissue culture protocol such that transgenics are recovered in sufficient number to warrant the choice of a particular regeneration technology (Christou, 1996). Embryogenic type II calli derived from immature embryos has been the most used tissue for the regeneration and transformation of maize (Frame et al., 2000; Gordon-Kamm et al., 1990). Recently, interest has been focused on using explants from mature seeds of cereals (Dai et al., 2001). For example, maize shoot tips and shoot meristematic cultures derived from mature seeds have been used as a target for particle bombardment transformation (Zhang et al., 2002; Zhong et al., 2003). Most recently, fertile maize plants have been regenerated using a new explant "split-seed" (Al-Abed et al., 2006). The split-seed technology is genotype independent and labor and space friendly, as no greenhouses are required to supply constant explant material. Hence the objectives in the present study were: (i) to utilize the split-seed regeneration technology and couple it with a transformation protocol to produce transgenic maize plants segregating for the CBF3 gene, and (ii) to assess the expression of CBF3 with respect to its ability to confer cold, drought, and salinity tolerance.
MATERIALS AND METHODS Nguồn mô cây Hạt của dòng R23 thu từ 1 công ty Pioneer Hi-Bred (Johnston, IA), các hạt được rửa bằng xà phòng diệt khẩn và khử trùng bề mặt bằng ethanol 70% trong 1 phút và xối nước 4 lần ngâm trong HgCl2 trong 7 phút. Các hạt rửa lần cuối với tia nước trong 24 giờ rồi cho nảy mầm trên môi trường MS(Murashige and Skoog, 1962) vitamin B5 bổ sun thêm9 µmol L–1 2,4-D (2,4-dichlorophenoxyacetic acid, Sigma, St. Louis, MO) trong 3 đến 4 ngày. Constructs Plasmid pC1301 chứa vùng không phiên mã intron ß-glucuronuridase (GUS) của Úc pCambia (Black Mountain, ACT, Australia) (Fig. 1A ). The GUS intron đã sử dụng để kiểm tra nhanh và xác định điều kiện biểu hiện trong bao mầm vòng bao mầm và mô phân sinh chồi. Figure 1. Maps of the constructs used for particle bombardment transformation: (A) pC1301 contains the GUS gene under 35S promoter; (B) pPDSG89 contains the CBF3 gene under the control of rd29A. CBF3 open reading frame (ORF) đã được khuyêch đại từ cây Arabidopsis (ecotype Columbia) genomic DNA by polymerase chain reaction (PCR) với vị trí Bam HI công thêm forward primer, 5'-GGATCCTCTAGATGAACTCATTTTCTGCTTTTTCTG-3', và vị trí Kpn I cộng thêm reverse primer, 5'-GGTACCTTTTAATAACTCCATAACGATACGTC- 3'. Sản phẩm PCR đã được nhân trong pBluescript SK(–) và đảm bảo chính xác trình tự. Một đoạn Hind III và BamHI của rd29A được nhân trong một vector pCambia sau đó, CBF3 ORF được giải phòng và hấp thu với BamHI và KpnI, nhân bổ sung trong cùngmột vector như phưonưg pháp của Kasuga et al. (1999). Câu trú này như minh họa ở hình B: pPDSG89 (Fig. 1B).
Explant Pretreatment and Particle Bombardment Sau khi tách hạt các mô được nuôi cấy 24 giờ trong môi trường tạo chồi multiple shoot induction medium (MSI) là môi trường MS cơ bản. Bổ sung thêm vitamin B5, 17.6 µmol L–1 BAP (6-benzylaminopurine, Sigma, St. Louis, MO), and 9.2 µmol L–1 kinetin (6- furfurylaminopurine, Sigma, St. Louis, MO) cộng thêm với : 1 mg L–1 glycine, 400 mg L–1 casein hydrolysate, 30 g L–1 sucrose, và 8 g L–1 agar đông cứng. pH điều chỉnh đến 5,8 trước khi cho thêm agar. Đặt 6 mô hạt đã tách trên một đĩa, xắp xếp với mặt cắt quay lên trên đối diện với bắn gen. Tất cả các đĩa đã bắn gen không đậy và đặt trong tủ hút 3 giờ để khô hạt Plasmid DNA được phân lập bằng HiSpeed Plasmid Midi Kit (Qiagen Sciences, Valencia, CA) và điều chnhr nông độ DNA đến 900 ng µL–1. Vi đạn vàng kích thước 0.6 µm in diameter (Bio-Rad Laboratories, Hercules, CA) được bọc 10 µL của plasmid DNA. Vỏ vàng vi đạn sau đó được trộn với 50 µL của 2.5 mol L–1 CaCl2 và 20 µL of 0.1 mol L–1 spermidine và xoay trong 20 min ở 4°C. Tiếp theo rửa 3 lần bằng ethanol 200 µL và ly tâm trong 1 phút sau mỗi lần rửa. Cuối cùng các vi đạn này được đưa vào 35 µL dung dịch ethanol và giữ trong đá. Tổng số 29 thí nghiệm đã thực hiện, xấp xỉ 3000 mô hạt, kiểm tra các thông số khác nhau : áp suất He (7.58, 9.31, và 10.69 MPa), khoảng cách mục tiêu (6 và 9 cm), số đạn (một hoặc hai). Tất cả thí nghiệm có GUS đối chứngvà 2 đối chứng âm tính : một viên có vỏ và một viên không Histochemical ß-Glucuronidase Assay Transient GUS expression in bombarded split-seed explants was visualized histochemically using X-Gluc (5-bromo-4-chloro-3-indolyl-ß-D-glucuronic acid, PhytoTechnology Laboratories, Shawnee Mission, KS) solution 48 h after bombardment, as described by Jefferson (1987). Bombarded split-seed explants testing different parameters were incubated in GUS solution, 10 explants per tube, for 24 h at 37°C. Transient GUS expression was then monitored under a stereomicroscope (Olympus SZX 12). Selection and Regeneration Twenty-four hours after bombardment, split-seed explants were transferred to MSI medium for 4 d in 16/8 h light/dark at 26°C as a recovery period. The explants were then transferred to a selection (MSI) medium supplemented with 25 mg L–1 hygromycin (Bioworld, Dublin, OH). The cultures were then subcultured biweekly on fresh medium three times before root induction. Putative transformants were separated and further cultured in rooting medium consisting of MS salts containing 3.2 µmol L–1 NAA (1-naphthaleneacetic acid, Sigma, St. Louis, MO) and supplemented with 10 mg L–1 hygromycin to ensure stringent selection before transferring to soil. Sau bắn 24 giờ các mô hạt được chuyển vào môi trường MSI nuôi cây trong 4 ngày ở nhiệt độ 26oC và 16/8 giờ sáng/tối là thời kỳ phục hồi. Các mô sau đó được chuyển đến môi trườngc họn lọc bổ sung thêm 25 mg L–1 hygromycin (Bioworld, Dublin, OH). Nuôi cây như vây một hai tuần trong môi trường sạch ba lần trước khi tạo rễ. các biến nạp
được phân tách để nuôi cấy trong môi truwngf tạo rễ gồm MS chưa 3.2 µmol L–1 NAA (1-naphthaleneacetic acid, Sigma, St. Louis, MO) và bổ sung thêm 10 mg L–1 hygromycin để đảm bảo chọnlọc nghiêm ngặt trước khi chuyển rangoài đất Molecular Analysis and Inheritance of CBF3 Gene in Progeny Genomic DNA được tách chiết từ 300 mg mô lá của các cây biến nạp cũng như đối chứng dạng dại. Sử dụng phương pháp cetyl trimethylammonium bromide cải tiến (Rogers và Bendich, 1985) để phân tích Southern blot( phương pháp xác định có mặt của DNA).. 10 µg của genomic DNA của mô biến nạp và dạng dại cũng như 50 pg của plasmid DNA được giải trình tự với Hind III hoặc KpnI (cắt tại một vị trí đơn trong plasmid) và số copy để ước tính số band lai trong phân tích Southern blot( phương pháp xác định có mặt của DNA).. Xắp xếp DNA được phân chia bằng điện di trên gel agarose 0,8%, và chuyển vào trong màng Hybond-N+ nylon (Amersham Biosciences, Little Chalfont, UK) according to Sambrook et al. (1989). Một đoạn 392 base pair (bp) PCR chứa CBF3 vùng mã hóa của pPDSG89 đã được khuyếch đại sử dụng cặp mồi oligonucleotide: forward 5'GAAACCGGCGGGTCGTAAGAAGTTT-3' và reverse 5'- TTTCGCTCTGTTCCGCCGTGTAAA-3'. Điều kienẹ khuyếch đại DNA như sau: mộth chu kỳ nhiệt 94°C trong 3 phút; 30 chu kỳ taị 94°C (45 s), 60°C (45 s), 72°C (2 min); và cuối cùng mở rộng tại 72°C (5 phút). DNA sau khi tinh sạch, gắn nhãn với [32P]-dCTP Để khẳng định di truyền của biến nạp ở các thế hệ tiếp theo, một cât To đã được lai trở lại với dòng R23. Các hạt thu được từ cây To trồng trong nhà kính và cho nảy mầm trên môi trường MS bổ sung thêm 25 mg L–1 hygromycin. Các cây T1 được kiểm tra để xác định di truyền của gen CBF3 theo phan tích Southern blot. Cac cây T1 tự phối tạo hạt để trồng thế hệ T2 được phân tích chọn lọc hygromycin và Southern blot ( phương pháp xác định có mặt của DNA). Reverse Transcription Polymerase Chain Reaction Analysis Phân tích PCR sao chép ngược Tổng RNA được phân lập từ các dòng CBF3 chuyển gen 7, 8, và 28 và đối chứng dạng dại gieo trồng tại 26°C trong tủ sinh trưởng, chất phản ứng thử Trizol (Invitrogen Life Technologies, Carlsbad, CA). RNA đượ xử lý DNase để loại trừ nhiễm bẩn DNA của RNA. Hai trăm nanograms của tổng số RNA được sử dụng để tổng hợp cDNA kit PCR sao chép ngược (one-shot reverse transcription polymerase chain reaction (RT-PCR) kit (Qiagen, Valencia, CA). Chương trình PCR như sau: 50°C trong 30 min; 94°C trong 15 min; 30 cycles gồm 94°C (45 s), 58°C (45 s), và 72°C (2 min); mở rộng cuối cùng 72°C trong 5 min. primers đặc thù sợi 638-bp cho sao mã CBF3 là: forward 5'-AACTCATTTTCTGCTTTTTCTGAA-3' and reverse 5'-TTAATAACTCCATAACGATACGTC-3'. As an internal contrNhư một đối chứng bên trong cho vùng mã hóa của ngô Actin là: forward 5'- TACAACGAGCTCCGTGTTTC-3' and reverse 5'- CTTTCTGACCCAATGGTGATG-3'. Các ản phẩm chạy điện di trên gel agarose 1%. Northern Blot Analysis Leaf tissues from transgenic plants and wild-type control plants that were treated with cold, drought, and salinity were frozen in liquid N2. Total RNA was isolated using Trizol reagent. The RNA was treated with DNase to eliminate false positives from DNA contamination of
the RNA. Fifteen micrograms of total RNA was loaded into a 1% formaldehyde gel and transferred onto a nylon membrane (Hybond-XL, Amersham Biosciences) according to a modified protocol from Sambrook et al. (1989). The blots were then hybridized with [32P]- dCTP-labeled probes at 65°C overnight and washed at high-stringency temperature (60°C). The CBF3 probe was prepared using specific primers flanking a 638-bp for the CBF3 transcripts: forward 5'-AACTCATTTTCTGCTTTTTCTGAA-3' and reverse 5'- TTAATAACTCCATAACGATACGTC-3'. A ZmCAT3 (Zea mays catalase 3; accession no. L05934) probe was prepared using specific primes flanking a 558-bp from the coding region: forward 5'-GCAACAACTTCCCCGTCTTCT-3' and reverse 5'- GCTGCTCGTTCTCGTTGAAGA-3'. The blots were stripped by boiling in 0.1% sodium dodecyl sulfate and rehybridized with a maize Actin probe prepared by using primers for the coding region of the maize Actin, which were: forward 5'- TACAACGAGCTCCGTGTTTC-3' and reverse 5'- CTTTCTGACCCAATGGTGATG-3'. Hybridized membranes were exposed to Kodak x-ray films at –80°C for 24 h. Stress Tolerance Studies Constant 10°C Experiments Thí nghiệm trong điều kiện 10oC ổn định Hạt T2 và dạng dạng gieo trong khay ( 36 hốc hạt/khay) Tổng số 236 hạt T2 và dạng dại ( 118 hạt mỗi loại Nảy mầm trong tủ sinh trưởng tại 26oC và 16/8 giờ sáng/tối Nhân T2 đại diện một quần thể biến nạp và một quần thể không biến nạp Cây con 5 ngày được xử lý 10oC ngày và đêm trong tủ sinh trưởng cũng dưới 16/8 giờ sáng/tối Các cây con được cung cấp đủ nước và dinh dưỡng Sinh trưởng và hình thái được đánh giá hàng tuần trong 4 tuần và theo dõi chiều cao cây Sau đó cây con chuyển ra trồng ở điều kiện bình thường. Theo dõi tỷ lệ sống sót và theo dõi sinh trưởng đến khi thành thục The T2 and wild-type seeds were sowed in Premier Pro-mix (Premier Horticulture, Quakertown, PA) in trays that hold 36 seeds per tray. A total of 236 T2 and wild-type seeds (118 each) were germinated in a growth chamber under 16/8 h light/dark at 26°C. The T2 generation represents a population of transgenic and segregated nontransgenic seeds. Five- day-old seedlings were challenged at 10°C day and night in a growth chamber also under 16/8 h light/dark. The seedlings were given an equal amount of water and fertilizer. Growth and morphology were monitored on a weekly basis for four weeks and the height of the plants was scored. The seedlings were then transferred to normal growth conditions for two weeks as a recovery period. The survival rate was calculated by dividing the number of surviving plants by the total number of seeds initially planted. Thirty T2 plants and the wild- type plants that survived were randomly selected for continued growth to maturity. The rate of fertility was scored as the number of partially or fully fertile plants divided by the number of selected plants. Cold and Freezing Tolerance (4, 0, and –2°C) The T2 seeds were germinated on MS medium containing 25 mg L–1 hygromycin to select for transgenic seeds. After 5 d, germinated seedlings were transferred to soil in trays and placed in a growth chamber under 16/8 h light/dark at 26°C. Wild-type seeds were germinated at the same time on MS medium without hygromycin and transferred to the
same type of trays containing soil. A total of 236 2-week-old transgenic and wild-type plants were used for each treatment—4, 0, and –2°C for 2 d, under 16/8 h light/dark—and were then returned to 26°C for two weeks. The number of surviving plants was counted and divided by the initial number of plants. Drought and Salinity Tolerance Four-week-old transgenic and wild-type plants were grown in 16.5-cm (6.5-inch) plastic pots (Kord Products, Toronto, ON, Canada) containing Premier Pro-mix (Premier Horticulture, Quakertown, PA) and kept in the greenhouse under 16/8 h light/dark at 26°C and 65% relative humidity. Drought stress was induced by withholding water for 7, 14, or 21 d. A total of 40 transgenic T2 and 40 wild-type plants were used for this study. The plants were monitored on a weekly basis for their phenotypes. The survival rate was scored from each water-withholding period after watering the treated plants and allowing a recovery period of two weeks. Đánh giá chịu hạn Cây biến nạp gen sau 4 tuần và dạng dại được trồng vào các chậu plastic kích thước 16,5cm chứa Premier Pro-mix (Premier Horticulture, Quakertown, PA) và giữa trong nhà kính tại 26oC và 16/8 giờ sáng/tối, độ ẩm 65%. Bất thuận hạn tạo ra bằng không tưới nước 7 , 14 hoặc 21 ngày. Tổng số 40 cây biến nạp T2 và 40 cây dạng dại sử dụng cho nghiên cứu chịu hạn Các cây được dánh giá hàng tuần trên cơ sở kểu hình, tỷ lệ sống sót và phục hồi sau hạn sau 2 tuần. For the salinity stress studies, two-week-old transgenic and wild-type plants grown in trays were subjected to 100, 200, and 400 mmol L–1 NaCl. A consistent volume of NaCl solution, irrespective of molarity, was added to the trays for one week. The plants were then given a two-week recovery period by watering regularly. The survival rate for each treatment was scored by dividing the number of surviving plants by the number of plants initially treated. Các cây biến nạp 2 tuần tuổi và cây dạng dại trồng trong các khay có nồng độ muối 100, 200, và 400 mmol L–1 NaCl. Một thể tích dung dịch NaCl phù hợp công thêm vào khay trong 1 tuần, các cây trồng sau đó được phục hồi bằng tưới đều đặn 2 tuần. Ghi nhận tỷ lệ sống sót của mỗi công thức bằng số cây sống sót so với số cây ban đầu khi đưa vào xử lý Electrolyte Leakage The determination of electrolyte leakage of plants treated with low and freezing temperatures and drought was conducted as described by Szalai et al. (1996), with some modifications. In the case of drought treatment, the ion leakage was estimated from 3, 7, and 14 d of withholding water, since none of the wild-type plants survived 21 d. Two-centimeter segments from the uppermost part of the leaf of stress-challenged plants were excised directly after each treatment, kept at room temperature for 24 h, and immersed in 20 mL of distilled water in flasks. The flasks were sealed and vortexed for 1 min, followed by 3 h of shaking at 75 rpm at room temperature. To test the level of electrolyte leakage due to low
and freezing temperatures and drought, the conductivity (C1) of the solution was measured using an electrical conductivity meter (Model HI 98312, Hanna Instruments, Ann Arbor, MI). The same leaf segments previously used to measure the initial electrolyte leakage (C1) were frozen at –80°C for 2 h, thawed for 1 h, and then immersed in the same solution to estimate the total potential ion leakage (C2). The relative percentage of injury due to stress was determined by dividing the mean ion leakage (C1) by the total leakage from frozen- killed samples (C2). Statistical Analysis For GUS expression analysis, the experimental unit was considered to be an explant with three replicates for each experiment. The mean number of GUS spots per explant was compared between each pressure point (covered vs. uncovered) and the number of shots (one or two). The data were analyzed by t-test at P = 0.05, using the SAS statistical analysis software, Version 9.1 (SAS Institute, Cary, NC). Phân tích biểu hiện của GUS với các mô cây, với thí nghiệm 3 lần nhắc lại, số vết trung bình của GUS/mô cây được so giữa mỗi điểm áp suất, phân tích t-test tại mức P = 0.05. Phana tích hồi quy, chi-square tại mức P = 0.05.Xác định khả năng chống chịu khác nhau giữa các cây biên nạp và dạng dại, 118 biến nạp vvà cùng một dạng dại, 3 lần lặp lại của 36 mẫu biến nạp sử dụng cho mỗi công thức bất thuận phân tchs t-test tại mức P = 0.05 sử dung phần mềm SAS, Version 9.1 For segregation analysis, the chi-square test at P = 0.05 was used to determine if deviations from expected ratios were within the acceptable range of variation using SAS, Version 9.1. To determine the difference in the stress tolerance between transgenic and wild-type plants, 118 transgenic and the same number of wild-type plants, from three replicates of 36, were used for each stress treatment. All percentage data from the survival rates and electrolyte leakage were transformed using arcsine transformation before statistical analysis. The data were analyzed by t-test at P = 0.05 to compare the means of transgenic and wild-type plants for each treatment, using SAS, Version 9.1. Table 1. Summary of the different parameters used for particle bombardment of split-seed explants that were cultured for 24 h on multiple-shoot induction medium and dried for 3 h before bombardment. Bảng 1: Tóm tắt các thông số khi bắn gen mô hạt đã được nuôi cây 24 giờ và làm khô trong 3 giờ trước khi bắn He pressure Distance¶ Shots Antibiotic- resistant shoots Explants bombarded CBF3 GUS Control no. MPa cm no. 83 12 12 7.58 6 1 0 76 12 12 7.58 9 1 2 85 12 12 9.31 6 1 1 97 12 12 9.31 9 1 0 87 12 12 7.58 6 2 1 80 12 12 7.58 9 2 2 80 12 12 9.31 6 2 4
80 12 12 9.31 9 2 3 90 12 12 10.69 6 1 2 76 12 12 10.69 9 2 4 90 12 12 10.69 9 2 2 CBF3 construct (pPDSG89). GUS construct (pC1301). RESULTS AND DISCUSSION Table 1. Summary of the different parameters used for particle bombardment of split-seed explants that were cultured for 24 h on multiple-shoot induction medium and dried for 3 h before bombardment. Optimization of Particle Bombardment Parameters for Split-Seed Explants Since different explants require different parameters for particle bombardment, we started by manipulating different parameters to optimize particle bombardment transformation protocols for split-seed explants. Split-seed explants were used for bombardment with gold particles coated with pC1301 (Fig. 1A), which contained a GUS gene to monitor the expression in bombarded regions of the split-seed explant. In the same experiment, the pPDSG89 plasmid was also used for CBF3 gene transformation (Fig. 1B). The explants were cultured on MSI medium for 24 h before bombardment, followed by an additional period of culturing after particle bombardment. The data for transgenic plants obtained from all of the experiments are summarized in Table 1 . Since cytokinins were identified as key mediators in plant cell division and cell cycles (Miller et al., 1955; Rediga et al., 1996), the incubation process may have altered the proliferation of targeted cells and made them more receptive to DNA uptake. Our results are consistent with those of Songstad et al. (1996), who found that immature maize embryos cultured for 2 to 4 d before bombardment were successfully transformed and regenerated into transgenic plants. Brettschneider et al. (1997) also reported that an increase in the transformation frequency was observed when immature embryos were cultured on callus induction medium before and after bombardment.
View this Table 1. Summary of the different parameters used for particle table: bombardment of split-seed explants that were cultured for 24 h on [in this multiple-shoot induction medium and dried for 3 h before bombardment. window] [in a new window] Osmotic pretreatment, or partial drying of the target cells before bombardment, has been proven to increase the frequency of successful transformation (Chen et al., 1998; Finer and McMullen, 1990). In many cases, mannitol or sorbitol is added to the medium as an osmotic inducer. Finer et al. (1999) reported that the osmotic treatment step prevented cell death, which occurs from wounding of the cell wall during microparticle penetration. In our experiments at 9-cm distance, we observed a significant difference in the average number of GUS spots from the cultures that were left uncovered in the laminar flow hood for 3 h (osmotic treatment) before bombardment and the covered cultures (P < 0.0001) with one shot using 7.58 and 9.31 MPa. There was no significant difference (P = 0.350), however, when 10.69 MPa was used in this treatment. When cultures were bombarded twice, there was a significant difference in the average number of GUS spots (P < 0.0001) at 7.58 MPa and no significant difference (P = 0.536 and P = 0.810) was observed at 9.31 or 10.69 MPa (Fig. 2 ). A decrease in target distance to 6 cm while keeping the pressure unchanged caused no changes in GUS expression. From these data, it may be concluded that the incidence of transient GUS expression is mainly influenced by pressure and the number of shots. Figure 2. Effect of drying split-seed explants before bombardment on transient GUS expression. Error bars represent the standard deviation. The means of GUS spots from each pressure point were analyzed by t-test (P = 0.05). View larger version (46K): [in this window] [in a new window] Selection and Regeneration of Transformants We determined that the optimum lethal dosage of hygromycin for untransformed split-seed explants was 25 mg L–1; therefore, this concentration was used to select for putative transformants (Fig. 3A and 3B). After bombardment, explants were given a recovery period
of four days on MSI medium before transferring to selection medium for a period of four to six weeks. The primary putative transformed shoots (Fig. 3C) that were regenerated during the first stage of selection were further subjected to a second stage of selection (Fig. 3D). In some cases, the putative shoots that needed further elongation before rooting were subjected to another round of selection for one week (Fig. 3E). A total of 21 antibiotic-resistant transformants were obtained by the end of all of the experiments, and 18 of the initial putative shoots were PCR positives for the CBF3 gene. Hence, the transformation frequency was estimated at 5%. Not all putative shoots elongated at the same rate and 15 plantlets whose growth was retarded were eliminated. Click on image to view larger version.
Figure 3. Production of transgenic maize plants using split-seed explant: (A) split-seed explants arranged for particle bombardment; (B) bombarded explants on selection medium; (C) shoot regeneration through first round of selection; (D) shoots after a second round of selection; (E) rooting of a CBF3 putative transformant; and (F) CBF3 transgenic plants grown in the greenhouse. Figure 3. Production of transgenic maize plants using split-seed explant: (A) split-seed explants arranged for particle bombardment; (B) bombarded explants on selection medium; (C) shoot regeneration through first round of selection; (D) shoots after a second round of selection; (E) rooting of a CBF3 putative transformant; and (F) CBF3 transgenic plants grown in the greenhouse. View larger version (99K): [in this window] [in a new window] Analysis of T0, T1, and T2 Plants Leaves from T0 plantlets that were hygromycin resistant were tested for the integration of
the CBF3 gene. Three lines that initially showed normal growth were confirmed positive for CBF3 by Southern blot analysis (Fig. 4A ). Southern blot analysis showed that Lines 7 and 8 had an estimated two to three copies of the CBF3 gene and they were sterile. In contrast, Line 28 had five copies but was fertile and went into further analyses. Các lá từ cây con To chịu với hygromycin đã kiểm tra phù hợp với gen CBF3, 3 dòng ban đầu biểu hienẹ sinh trưởng bình thường khẳng định dương tính với CBF3 qua phân tích Southern blot như hình điện di, dòng 7 và 8 đánh giá có 2 – 3 bản sao của gen CBF3 và chúng cằn cỗi, dòng 28 có 5 bản sao gen nhưng rất tốt khi đưa vào những phana tích tiếp theo Figure 4. Southern blot analysis of T0, T1, and T2 CBF3 plants: (A) Southern blot analysis of T0 plants, showing Lane 1—kb ladder; Lane 2—untransformed plant digested with KpnI; Lanes 3, 5, and 7—Lines 7, 8, and 28 digested with Hind III; Lanes 4, 6, and 8— Lines 7, 8, and 28 digested with KpnI; and Lane 10— plasmid positive control digested with KpnI; (B) T1 plants that originated from Line 28, showing Lane 1— View larger version (67K): kb ladder, Lane 2—untransformed plant; Lanes 3 to 9 —plants no. 1, 3, 4, 7, 11, 13, and 14 digested with [in this window] KpnI; and Lane 11—plasmid positive control digested [in a new window] with KpnI; (C) T2 plants digested with KpnI, showing Lane 1—kb ladder; Lane 2—untransformed plant; Lanes 3 to 20—T2 plants no. 1 to 18; and Lane 21— plasmid positive control.
Figure 4. Phân tích Southern blot các cây CBF3 T0, T1, và T2 CBF3: (A) Các cây T0, biểu hiện tháng Lane 1—; Lane 2—cây không biến nạp xếp với KpnI; Lanes 3, 5, và 7—các dòng 7, 8, và 28 xếp với Hind III; Lanes 4, 6, and 8—các dòng 7, 8, và 28 xếp với KpnI; và Lane 10—plasmid đối chứng dưonưg tính xếp với KpnI; (B) Các cây T1 nguồn từ dòng 28, biểu hiện thang Lane 1—kb, Lane 2—cây không biến nạp; Lanes 3 to 9—các cây số 1, 3, 4, 7, 11, 13, và 14 xếp với KpnI; và Lane 11—plasmid đối chứng dưonưg tính xếp với KpnI; (C) Các cây T2 xếp với KpnI, biểu hiện Lane 1—kb ladder; Lane 2—cây không biến nạp; Lanes 3 to 20— các cây T2 số 1 to 18; và Lane 21—plasmid dương tính đối chứng. Dòng 28 cho thụ phấn chéo với R23 tạo ra con cái T1. Phana tích riêng rẽ cả hai là marker hpt (hygromycin phosphotransferase) và gen CBF3 trong các cây T1 thực hienẹ bằng cho hạt nảy mầm trên môi trường MS bổ sung thêm mg L–1 hygromycin. Phân tích Southern blot biểu hiện 5 bản sao gen CBF3, kết quả 91 cây T1 phân ly theo tỷ lệ 1:1 (P = 0.905). Điều này có thể giải thích rằng 5 gen cùng insert vào một lô cút. Ngoài ra các cây cây này còn biểu hiện sinh trường bình thường như hinh 3F Line 28 was outcrossed to R23 to produce T1 progeny. Segregation analysis of both the selectable marker hpt (hygromycin phosphotransferase) and the CBF3 gene in T1 plants was performed by germinating the seeds on MS medium supplemented with 25 mg L–1 hygromycin (Table 2 ). As expected and despite the fact that the Southern blot analysis unambiguously showed five copies of CBF3, the resulting 91 T1 plants segregated CBF3 in a 1:1 ratio (P = 0.905). This argues that it is probable that the five genes inserted at a single chromosomal locus. In addition, these plants showed normal growth morphology (Fig. 3F) and were further confirmed for the transmission of CBF3 by Southern blot analysis (Fig. 4B). Southern blot analysis of Line 28 and the tested T1 progeny plants showed the same integration pattern with multiple hybridization bands (Fig. 4A and 4B). Dai et al. (2001) also showed that most of the fertile transgenic plants from particle bombardment segregated GUS and hpt genes in a Mendelian fashion. View this Table 2. Segregation analysis of T1 and T2 plants for the CBF3 gene. table: Segregation ratios 1:1 or 3:1 depend on crosses (P = 0.05). [in this window] [in a new window]
Transgenic T1 plants were self-pollinated to produce T2 progeny and the segregation analysis of marker hpt and CBF3 in the T2 plants gave a 3:1 ratio (P = 0.871) also for the presence vs. absence of both genes (Table 2). The inheritance of CBF3 in T2 plants was also confirmed by Southern blot analysis (Fig. 4C). Table 2. Segregation analysis of T1 and T2 plants for the CBF3 gene. Segregation ratios 1:1 or 3:1 depend on crosses (P = 0.05). 2 Seeds tested Generation Segregation for hygromycin and CBF3 no. 91 T1 44:47 0.20 NS 113 T2 87:26 0.27 NS T0 plant was crossed with untransformed plants to produce T1 seeds; T1 plants were self- pollinated to produce T2 seeds. Segregation estimation was collected based on germination on selection (hygromycin) medium (germinated/ungerminated) and Southern blot analysis for CBF3. NS, not significant at P = 0.05. CBF3 Expression The expression level of CBF3 was detected in transgenic lines 7 and 8, even under normal growth conditions (26°C), and the plants showed a slight stunted growth and were sterile. In contrast, the expression level in Line 28 was hardly detected in RT-PCR analysis at normal growth temperature and resulted in a fully fertile plant with normal phenotype (Fig. 5 ). Overall, no differences in morphology or growth were observed between the stable transgenic plants expressing the CBF3 gene and wild-type plants under normal growth conditions. Pellegrineschi et al. (2004) observed nonuniform germination in rd29A:CBF3 transgenic wheat seeds from normal and stressed conditions but the plants recovered and showed normal growth and morphology in later stages. Mức biểu hiện của CBF3 được phát hiện trong các dòng biến nạp gen 7 và 8, ngay cả dưới điều kienẹ sinh trưởng bình thường 26oC và các cây biểu hiện ức chế sinh trưởng nhẹ (hơi cằn cỗi) và bất thụ. Ngược lại dòng 28 được phát hiện trong phân tích RT-PCR (Reverse transcription polymerase chain reaction analysis) tại nhiệt độ sinh trưởng bình thường và cho sinh trưởng bình thường, kiểu hình hoàn chỉnh. Nhìn chung không có sự khác biệt về kiểu hình và sinh trưởng quan sat ở các cây biến nạp CBF3 ổn định và các cây dạng dại dướ điều kiện sinh trưởng bình thường
Figure 5. Reverse transcription polymerase chain reaction analysis of CBF3 gene expression in T0 Lines 7, 8, and 28 grown at 26°C; C is a wild-type plant, maize actin was used as a loading control. Figure 5. Reverse transcription polymerase chain reaction analysis of CBF3 gene expression in T0 Lines 7, 8, and 28 grown at 26°C; C is a wild-type plant, maize actin was used as a loading control. View larger version (38K): [in this window] [in a new window] A high expression level of targeted stress-inducible genes was observed in transgenic plants when CBF3 was driven by the CaMV 35S promoter and those plants showed significant tolerance to drought, freezing, and high-salt stresses (Gilmour et al., 2000; Liu et al., 1998).
Transgenic plants with CBF3 under CaMV 35S, however, showed growth retardation under normal growth conditions (Kasuga et al., 2004). Use of the stress-inducible rd29A promoter instead of the constitutive CaMV 35S promoter for the overexpression of CBF3 minimized the negative effects on plant growth in transgenic Arabidopsis (Kasuga et al., 1999). To monitor the expression profile of CBF3 transcripts under stress conditions, plants were grown at 26°C and were then first subjected to cold temperatures gradually, with a duration of 3 h for each period. Northern blot analysis indicated that CBF3 transcripts were first detected at 20°C and the amount of mRNA corresponding to CBF3 was increased to reach the highest level at 4, 0, and –2°C (Fig. 6 ). Zarka et al. (2003) reported that the transcript levels of the CBF genes were detected at 14°C when Arabidopsis plants were subjected to gradually lower temperatures. Các cây biến nạp mang gen CBF3 dưới CaMV 35S biểu hiện trở lại sinh trưởng bình thường (Kasuga et al., 2004). Sử dụng đoạn hoạt hóa rd29A gây bất thuận thay thế cho CaMV 35S để mở rộng biểu hiện của CBF3 ảnh hưởng nhỏ nhất đến cây Arabidopsis biến nạp gen(Kasuga et al., 1999). Để kiểm tra biểu hieựen của CBF3 dưới điều kiện bất thuận. Các cây được trồng ở 26oC và sau đó xử lý lạnh với thời gan 3 giờ cho mỗi mức nhiệt độ. Phân tchs Northern blot chỉ ra rằng phiên mã BCF3 được phát hienẹ ở 20oC và số lưonựg mRNA phù hợp với sự tăng của CBF3 đạt mức cao nhất tại 4 và 0 và -2oC Figure 6. Northern blot analysis of CBF3 transcripts after T2 plants derived from Line 28 were treated with different low temperatures. Plants were grown at 26°C and then were gradually exposed to low temperatures (20, 16, 12, 10, 4, 0, and –2°C), 3 h for each temperature. Full length of the coding region of CBF3 was used as a probe and actin was used as a loading control.
Figure 6. Northern blot analysis of CBF3 transcripts after T2 plants derived from Line 28 were treated with different low temperatures. Plants were grown at 26°C and then were gradually exposed to low temperatures (20, 16, 12, 10, 4, 0, and –2°C), 3 h for each temperature. Full length of the coding region of CBF3 View larger version (52K): was used as a probe and actin was used as a loading control. [in this window] [in a new window] Analyses of CBF3 Plants Cold and Freezing Tolerance We conducted different studies to examine whether CBF3 transgenic plants will be more tolerant to low and freezing temperatures than the wild-type plants. Five-day-old seedlings from T2 and wild-type seeds that were grown in trays at 26°C were transferred to a constant 10°C growth chamber for four weeks under 16/8 h light/dark (Fig. 7A ). After one week, wild-type seedlings began to show injury symptoms and turned yellow, and growth had stopped. In comparison, transgenic seedlings continued to grow at 10°C and remained green (Fig. 7B). By the end of four weeks, 80% of the wild-type seedlings were dead, but transgenic seedlings were more tolerant and survived the 10°C four-week period (Fig. 7C and 7D). After all of the plants were moved to the greenhouse and kept at 26°C for a two- week recovery period,
when kept at 10°C: (A) 5-d-old seedlings grown at 26°C (normal condition); (B) 1-wk-old seedlings; and (C) 4-wk-old seedlings. (D) Mean heights of transgenic and wild-type seedlings at different time periods, with error bars representing the standard deviation; and (E) survival and fertility rates of transgenic and wild-type plants after a 2-wk recovery period from 10°C in the greenhouse, during which surviving plants were grown to maturity. Figure 7. Comparison of growth characteristics, survival rate, and fertility rate between transgenic T2 seedlings derived from Line 28 (left tray) vs. wild- type seedlings (right tray) when kept at 10°C: (A) 5-d- old seedlings grown at 26°C (normal condition); (B) 1-wk-old seedlings; and (C) 4-wk-old seedlings. (D) Mean heights of transgenic and wild-type seedlings at View larger version (54K): different time periods, with error bars representing the [in this window] standard deviation; and (E) survival and fertility rates [in a new window] of transgenic and wild-type plants after a 2-wk recovery period from 10°C in the greenhouse, during which surviving plants were grown to maturity. The expression of CBF3 was also monitored at lower temperatures, and the transcript levels were analyzed by Northern blot analysis. When transgenic plants were subjected to cold shock at 4°C, the plants were moved directly from 26°C into 4°C growth chambers for a 24- h period; transcripts of the CBF3 gene were detected by Northern blot analysis after plants were exposed to 4°C for 1 h and the expression level increased after 3 h of exposure and was gradually reduced after 5, 7, 12, and 24 h (Fig. 8A ). In contrast, the transcripts were observed within 15 min of exposure to 0°C and continued to be expressed for the 24-h period (Fig. 8B). To determine whether the CBF3 transgenic plants are resistant to lower (4°C) and perhaps freezing temperatures (0 and –2°C), two-week-old transgenic and wild- type plants grown in trays at 26°C were transferred to 4, 0, and –2°C conditions for 48 h and returned to the greenhouse at 26°C. Directly following the treatments, we observed that all of the wild-type plants wilted, whereas transgenic plants maintained their healthy morphology (Fig. 9A, 9B, and 9C ). We then performed the electrolyte leakage test to evaluate the damage that could have occurred to the cell membrane of the transgenic and wild-type plants as a result of exposure to cold and freezing temperatures. Two-centimeter segments from the upper leaf tip of treated plants were used for the electrolyte leakage test. For the wild-type plants, the ion leakages were 42.8, 78, and 83.9% at 4, 0, and –2°C, respectively, compared with transgenic plants at 11.4, 24.8, and 25.9%, respectively (Fig. 9G). These results clearly demonstrate that the ion leakage of the plants segregating CBF3 was significantly reduced after cold and freezing treatments when compared with control plants (P < 0.0001). To further evaluate the tolerance of transgenic and wild-type plants to the 48-h treatments at 4, 0, and –2°C, the survival rate was also calculated after a two-week recovery period in the greenhouse (Fig. 9D, 9E, and 9F). While 57.4% of the wild-type
plants survived the 4°C treatment, 89.2% of the transgenic plants endured. The survival rate percentage of the wild-type plants continued to drop significantly from 0 and –2°C treatments (P < 0.0001), an estimated 40 and 15.7%, respectively, compared with the transgenic plants survival rates of 79.1 and 73.4%, respectively (Fig. 9H). Figure 8. Northern blot analysis of T2 plants derived from Line 28 that were exposed to 4 and 0°C for different periods of time: (A) accumulation of CBF3 transcripts in response to exposure to 4°C for 1, 3, 5, 7, 12, and 24 h (C is a wild-type plant and TN is a transgenic (T2) plant derived from Line 28 grown at 26°C); (B) T2 plants derived from Line 28 that were exposed to 0°C for 15 min, 30 min, and 1, 3, 6, 12, and 24 h (C is a wild-type plant); actin was used as a loading control.