摘要
本研究通过诱导不同基因型海岛棉下胚轴的体细胞胚胎发生,进一步优化海岛棉再生体系并且获得再生植株,并以新海30号胚性愈伤组织为受体材料,通过农杆菌介导法转化NAC转录子家族,获得转基因再生植株。为今后新疆海岛棉抗旱分子育种奠定了基础。本研究取得的主要结果如下：
1.通过愈伤诱导时期激素的配比,IBA含量的不同和糖源的不同对海岛棉胚性愈伤组织分化能力的影响,活性炭对再生苗生根的影响,进一步优化海岛棉的植株再生体系。结果表明愈伤诱导的最佳激素配比为0.1 mg/L2,4-D+0.1 mg/L KT;胚性愈伤诱导率最佳的培养基是加入含量为0.05 mg/L的IBA,糖源为30 g/L葡萄糖；对再生苗生根的效果最好的是培养基中加入0.1%的活性炭。
2.为了进一步筛选优化的根癌农杆菌介导海岛棉外源基因转化体系,提高海岛棉外源基因转化率。以生理状态基本一致的海岛棉新海30号的胚性愈伤组织为受体材料,以绿色荧光蛋白基因(GFP)作为报告基因,将转化的时间和共培养条件作为试验要素,筛选最适宜的浸染时间和共培养条件范围。结果表明,以GFP为报告基因进行根癌农杆菌介导的海岛棉外源基因转化体系的最佳浸染时间为15 mmin,共培养时间为24 h。
3.农杆菌介导海岛棉新海30号的胚性愈伤组织,获得转NAC转录子家族基因的转基因植株。转化条件为：新海30号胚性愈伤组织预处理10 d后,于OD值为0.5的菌液中浸染15 mmin后共培养24 h。处理完毕后,将胚性愈伤组织转入添加50 mg/L卡那霉素及400 mg/L头孢霉素的培养基中诱导转基因体细胞胚胎分化,可获得15%左右的转基因体细胞胚胎,其中5%可获得正常再生植株。通过PCR检测,初步证明NAC转录子家族基因中的NAC2、NAC3、NAC6已转化到再生的棉花植株中。
This study is further to optimize plant regeneration system of the Sea Island cotton and obtain regenerated plants by inducing somatic embryogenesis of Sea Island cotton hypocotyl of different genotypes; And with the embryogenic callus as receptor, transform NAC transcripts family with agrobacterium mediation and obtain transgenic plant, in order to lay the foundation for molecular breeding of drought-resistance. The major results are as follows:
1. By exploring effects of the ratio of hormones, different IBA contents and different carbohydrate sources to differentiatial ability of Sea Island cotton embryogenic callus, activated carbon contents on the rooting of regenerated plants, further to optimize plant regeneration system of the Sea Island cotton. The results showed that the best hormones ratio was 0.1 mg/L 2,4-D+0.1 mg/L KT, the best media was 0.05 mg/L IBA,30 g/L glucose and 0.1% activated carbon.
2. To further screen optimized agrobacterium-mediated gene transfer system for raising transformation efficiency of exogenous gene in Sea Island cotton, appropriate time of infection and co-cultivation had been confirmed by treating embryogenic callus of Xinhai 30 with the same physiological state and GFP gene respectively as receptor materials and reporter gene, and the time of transformation and co-cultivate were treated as experimental factors. The results indicated that the most suitable time of infection and co-culivate for agrobacterium-mediated gene transfer system of Sea Island cotton was 15 min and 24 h, respectively.
3. Transgenic plants of NAC transcripts family were obtained by embryogenic callus of Sea Island cotton Xinhai 30 using the agrobacterium-mediated method. Embryogenic callus of Xinhai 30 were pretreated 10 d, co-culture 24 h after soaking 15 min in the bacilli of OD value was 0.5. After that, transferring the embryogenic callus to media contained 50 mg/L kanamycin and 400 mg/L cephamycin for inducing differentiation of somatic embryos, which was able to obtain 15% transgenic somatic embryos, and 5% of which enabled to obtain normal regenerated plants. PCR tests showed that NAC2、NAC3、NAC6 of NAC Transcripts Family had been transferred into regenerated cotton plants.
引文
[1]刘毓湘.当代世界棉业[M].中国农业出版社,1995.
[2]黄滋康.中国棉花品种及其系谱[M].中国农业出版社,2007.
[3]Benedict J, Sachs E, Altman D.Field performance of cottons expressing transgenic CrylA insecticidal proteins for resistance to Heliothis virescens and Helicoverpa zea (Lepidoptera:Noctuidae)[J]Journal of economic entomology,1996,89 (1):230-238.
[4]阳立恒.新疆棉花再生体系的建立及其遗传转化影响因素的研究[D].乌鲁木齐：新疆农业大学硕士学位论文,2008.
[5]Haberlandt GPhysiological plant anatomy.[M].London,1914.
[6]段肖霞.甜菜离体培养直接器官发生及遗传转化的研究[D].乌鲁木齐：新疆农业大学硕士学位论文,2007.
[7]武秀明,刘传亮,张朝军,等.棉花体细胞胚胎发生的研究进展[J].植物学通报,2008,25(004)：469-475.
[8]Bayes M, Price M.An Essay towards solving a Problem in the Doctrine of Chances. [J].Philosophical Transactions,1979,53:370.
[9]Davidonis GH, Hamilton RH.Plant regeneration from callus tissue of Gossypium hirsutum L[J].Plant science letters,1983,32 (001):89-93.
[10]贺雅婷.海岛棉再生体系的建立及SATI基因遗传转化初步研究[D].乌鲁木齐：新疆农业大学硕士学位论文,2008.
[11]董合忠,陈志贤.棉花细胞组织培养的调控研究[J].莱阳农学院学报,1990,7(001)：28-33.
[12]陈志贤,等.利用农杆菌介导法转移tfdA基因获得可遗传的抗2,4-D棉株[J].中国农业科学,1994,27(002)：31-37.
[13]张宝红,李秀兰,等.棉花茎尖分生组织培养再生植株[J].中国棉花,1993,20(004)：13-13.
[14]张宝红,刘方,刘志红,等.棉花优良品种中棉所19高频体细胞胚胎发生细胞系筛选与植株再生[J].作物学报,2000,26：2-8.
[15]谢德意,金双侠,郭小平,等.长江和黄河流域棉区棉花品种体细胞胚胎发生和植株再生比较研究[J].作物学报,2007,33(03)：394-400.
[16]贺雅婷,曲延英,孔庆平,等.海岛棉愈伤组织诱导及分化的影响因素初探[J].分子植物育种, 2008,6(003)：597-602.
[17]赖来,潘俊松,何欢乐,等.农杆菌介导的MADS-box基因转化黄瓜初步研究[J].上海交通大学学报：农业科学版,2007,25(004)：374-382.
[18]白云凤,杨红春,曲琳,等.抗甘蔗花叶病毒的无标记反向重复转基因玉米[J].作物学报,2007,33(006)：973-978.
[19]严勇亮,曲延英,代鑫,等.新疆和黄河流域棉区棉花品种体细胞胚胎发生和植株再生比较研究[J].新疆农业科学,2010,(001)：14-19.
[20]张天真,唐灿明,朱协飞,等.转基因抗虫杂交棉南抗3号的选育及应用[J].CHINA COTTON,2001,28(10)：254-268.
[21]欧阳立明,吴宏文,喻子牛,等.害虫对转Bt基因植物抗性的治理策略[J].农业科学,2001,28(2)：125-136.
[22]郭三堆,倪万潮.双价抗虫转基因棉花研究[J].中国农业科学,1999,32(003)：1-7.
[23]Bowler C, Montagu MV, Inze D.Superoxide dismutase and stress tolerance[J].Annual Review of Plant Biology,1992,43 (1):83-116.
[24]Karpinski S, Reynolds H, Karpinska B.Systemic signaling and acclimation in response to excess excitation energy in Arabidopsis[J].Science,1999,284 (5414):654-664.
[25]McKersie BD, Chen Y, de Beus M.Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.) [J].Plant Physiology,1993,103 (4):1155-1162.
[26]Gupta A S, Webb R P, Holaday A S,et al.Overexpression of superoxide dismutase protects plants from oxidative stress (induction of ascorbate peroxidase in superoxide dismutase-overexpressing plants)[J].Plant Physiology,1993,103 (4):1067-1075.
[27]Allen R D.Dissection of oxidative stress tolerance using transgenic plants[J].Plant Physiology, 1995,107 (4):1049-1058.
[28]Allen R, Trolinder N.Expression of superoxide dismutase in transgenic plants leads to increased stress tolerance[J].Beltwide Cotton Conferences (USA),1995,204:1237-1245.
[29]Landfald B, Strom A.Choline-glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli[J] Journal of bacteriology,1986,165 (3):849-864.
[30]Lilius G, Holmberg N, Blow L.Enhanced NaCl stress tolerance in transgenic tobacco expressing bacterial choline dehydrogenase[J].Nature Biotechnology,1996,14 (2):177-180.
[31]Tarczynski MC, Jensen RG, Bohnert HJ.Stress protection of transgenic tobacco by production of the osmolyte mannitol[J].Science,1993,259 (5094):508-515.
[32]秦红霞,宋玉霞,刘敬梅,等.植物DREB转录因子的研究进展及应用[J].生物技术通讯,2006,17(005)：780-783.
[33]彭辉,麻浩,等.植物NAC转录因子家族研究概况[J].植物学报,2010,45：236-248.
[34]Umbeck P, Johnson G, Barton K, et al.Genetically transformed cotton (Gossypium hirsutum L.) plants[J].Nature Biotechnology,1987,5 (3):263-266.
[35]Firoozabady E, DeBoer DL, Merlo DJ, et al. Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants[J].Plant Molecular Biology,1987, 10 (2):105-116.
[36]Bayley C, Trolinder N, Ray C, et al. Engineering 2,4-D resistance into cotton[J].TAG Theoretical and Applied Genetics,1992,83 (5):645-649.
[37]郭三堆,张秀梅,崔洪志,等.抗蚜虫兼抗除草剂的转基因棉花研究[J].云南大学学报：自然科学版,1999,21(3)：127-130.
[38]焦改丽,李俊峰.利用新的外植体建立棉花高效转化系统的研究[J].棉花学报,2002,14(001)：22-27.
[39]李晓,王学德,朱玉贤,等.农杆菌真空渗透法转化花粉获得棉花转基因植株[J].作物学报,2002,28(5)：245-257.
[40]Balasubramanian S, Konana P, Menon NM. Customer satisfaction in virtual environments:A study of online investing[J].Management Science,2003:871-889.
[41]Bakhtiari S, Safavi-Naini R, Pieprzyk J.Cryptographic hash functions:A survey[J].Plant Cell Reports,1995:987-997.
[42]金双侠,张献龙,聂以春,等.启动子诱捕在棉花基因组中的功能分析[J].遗传学报,2005,32(012)：1266-1274.
[43]陈英,黄敏,王明庥,等.植物遗传转化新技术和新方法[J].中国生物工程杂志,2005,25(9)：94-98.
[44]吕秀娟.新疆陆地棉转化雪莲磷脂酰乙醇胺结合蛋白基因的研究[D].乌鲁木齐：新疆农业大学硕士学位论文,2007.
[45]饶慧斌.棉花远缘杂交育种研究现状及前景分析[D].陕西杨凌：西北农林科技大学硕士学位论 文,2006.
[46]Sanford J C, Wolf E D, Allen N K.Method for transporting substances into living cells and tissues and apparatus therefor[J].Google Patents,1990:346-358.
[47]Finer J J, McMullen M D.Transformation of cotton (Gossypium hirsutum L.) via particle bombardment[J].Plant Cell Reports,1990,8 (10):586-589.
[48]谢迎秋,朱祯,等.棉花曲叶病毒反式作用因子AC2的功能初探[J].植物学报,2001,43(005)：517-521.
[49]董云洲,贾士荣,等.基因枪在植,物遗传转化上的应用[J].中国生物工程杂志,1993,13(2)：15-18.
[50]叶兴国,徐惠君.小麦农杆菌介导转基因植株的稳定获得和检测[J].中国农业科学,2001,34(5)：469-474.
[51]周光宇,翁坚,龚蓁蓁,等.农业分子育种授粉后外源DNA导入植物的技术[J].中国农业科学,1988,21(3)：1-6.
[52]黄骏麒,钱思颖,刘桂玲,等.外源海岛棉DNA导致陆地棉性状的变异[J].遗传学报,1981,8(1)：56-62.
[53]龚蓁蓁,沈慰芳,周光宇,等.授粉后外源DNA导入植物技术通过花粉管通道进入胚囊[J].中国科学,1988,6：611-614.
[54]谢道昕,范云六,倪丕冲,等.苏云金芽孢杆菌杀虫基因导入中国栽培水稻品种中花11号获得转基因植株[J].中国科学,1991,21(8)：830-834.
[55]吴小月.外源DNA导入技术在棉种改良中的应用研究[J].湖南农业大学学报(自然科学版),1993,1：24-35.
[56]张红艳,李冠,李仁敬,等.彩色棉再生体系影响因子及抗病基因NP-1转化的研究[J].新疆大学学报：自然科学版,2004,21(001)：94-98.
[57]郭江勇,王义琴,吴明刚,等.棕色棉和绿色棉遗传多样性的比较研究[J].遗传学报,2004,26(001)： 63-68.
[58]侯文胜,郭三堆,路明,等.基因枪法获得转基因小麦植株[J].中国农业科学,2003,36(5)：469-472.
[59]欧阳杰.内切-1,4-β-葡聚糖酶基因转化棉花及转基因植株的再生研究[D].长沙：湖南农业大学硕士学位论文,2007.
[60]朱生伟,黄国存,孙敬三,等.外源DNA直接导入受体植物的研究进展[J].植物学通报,2000,17(1)：11-16.
[61]Jackson S, Zhang P, Chen W, et al.High-resolution structural analysis of biolistic transgene integration into the genome of wheat[J].TAG Theoretical and Applied Genetics,2001,103 (1): 56-62.
[62]Rutgers E, Ramulu K, Dijkhuis P, et al. Identification and molecular analysis of transgenic potato chromosomes transferred to tomato through microprotoplast fusion[J].TAG Theoretical and Applied Genetics,1997,94 (8):1053-1059.
[63]Sato Y, Tamaoki M, Matsuoka M, et al. Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSHl lead to altered morphology of leaves in transgenic tobacco[J].Molecular and General Genetics,1996,251 (1):13-22.
[64]Pedersen C, Zimny J, Becker D, et al. Localization of introduced genes on the chromosomes of transgenic barley, wheat and triticale by fluorescence in situ hybridization[J].Theoretical and Applied Genetics,1997,94 (6):749-757.
[65]巩振辉.CaMV基因在拟南芥上的遗传转化及交义保护[J].西北农业大学学报,1997,25(004)：6-12.
[66]邓晓东,费小雯,等.萝卜抗真菌蛋白基因AFPs在大肠杆菌中的表达及其转化番茄的研究[J].园艺学报,2001,28(004)：361-363.
[67]李荣田,张忠明.RHL基因对粳稻的转化及转基因植株的耐盐性[J].科学通报,2002,47(008)：613-617.
[68]翟文学,李晓兵,田文忠,等.由农杆菌介导将白叶枯病抗性基因Xa21转入我国的5个水稻品种[J].遗传学报,2000：345-356.
[69]张云孙,李援亚,宋敏,等.导入AGPase基因的转基因可育水稻及其经济性状的研究[J].华北农学报,2001,4：47-58.
[70]胡军,王静,卢新亚,等.病原性真菌PCR检测方法的建立[J].郑州大学学报(医学版),2004,39(2)：124-136.
[71]陈秀花,刘巧泉,王宗阳,等.反义Wx基因导入我国籼型杂交稻重点亲本[J].遗传学报,2002,47(9)： 76-89.
[72]高越峰,荆玉祥.高赖氨酸蛋白基因导入水稻及可育转基因植株的获得[J].植物学报,2001,43 (005)：506-511.
[73]安韩冰,朱祯.基因枪法转化水稻获得可育的转抗虫基因水稻再生植株[J].高技术通讯,2001,11(002)：12-16.
[74]严蔚东,王校常,何锶洁,等.利用外源钾通道基因改良水稻钾素营养[J].中国水稻科学,2002,16(1)：77-79.
[75]戴顺洪,李良材,丁月云,等.水稻基因枪法多基因转化研究[J]Acta Genetica,1998,25(4)：345-350.
[76]简玉瑜,董春.应用基因枪将蚕抗菌肽基因导入水稻获抗白叶枯病株系[J].华南农业大学学报,1997,18(004)：1-7.
[77]许新萍,张建中.抗稻瘟病和纹枯病的转基因水稻新品系[J].中山大学学报：自然科学版,2001,40(003)：131-132.
[78]Jani D, Meena LS, Rizwan-ul-Haq QM, et al.Expression of cholera toxin B subunit in transgenic tomato plants[J].Transgenic research,2002,11 (5):447-454.
[79]陈青,薛朝阳,吴俊杰,等.烟草花叶病毒移动蛋白基因转化烟草及在转基因烟草中的表达[J].西北植物学报,2002,35(5)：435-347.
[80]张荃,张慧.HAL1基因转化番茄及耐盐转基因番茄的鉴定[J].生物工程学报,2001,17(006)：658-662.
[81]Djennane S, Chauvin J E, Quiller, et al.Introduction and expression of a deregulated tobacco nitrate reductase gene in potato lead to highly reduced nitrate levels in transgenic tubers[J].Transgenic research,2002,11 (2):175-184.
[82]朱飞舟.转抗病基因水稻的分子检测及农艺性状评价[D].长沙：湖南农业大学硕士学位论文,2004.
[83]王清连,刘方.棉花组织培养直接胚胎发生和植株再生[J].棉花学报,2002,14(006)：340-343.
[84]商海红,刘传亮,张朝军,等.棉花体细胞胚发生机理的研究进展[J].西北植物学报,2009,29(003)：637-642.
[85]周小梅,赵云云.转MDMV CP基因玉米植株的再生[J].植物研究,2006,26(4)：234-238.
[86]张宝红,刘方.外源激素对棉花体细胞胚胎发生及发育的调控作用[J].棉花学报,2000,12(001)：17-21.
[87]张宝红,李秀兰,李付广,等.棉花组织培养中畸形胚的发生和转化[J].作物学报,1996,22(1)： 107-111.
[88]董合忠,焦改丽.2,4-D,KT棉花愈伤组织的诱导和体细胞胚胎发生的影响[J].华北农学报,1993,8(003)：87-91.
[89]Finer JJ.Plant regeneration from somatic embryogenic suspension cultures of cotton (Gossypium hirsutum L.)[J].Plant Cell Reports,1988,7 (6):399-402.
[90]Trolinder N L, Xhixian C.Genotype specificity of the somatic embryogenesis response in cotton[J].Plant Cell Reports,1989,8 (3):133-136.
[91]张宝红,李国良,迟文峰,等.紫花苜蓿耐盐生理的初步研究[J].现代农业科技,2006,12：6-7.
[92]师海荣.陆地棉愈伤组织诱导及体细胞胚发生的生化机制研究[J].中国农学通报,2007,23(2)：77-80.
[93]张文胜,张宝红.糖源在棉花愈伤组织诱导胚胎发生与发育中的作用[J].江西农业大学学报,1997,19(003)：34-39.
[94]吴慎杰,李飞飞,张天真,等.插皮接技术在棉花再生植株嫁接中的应用[J].棉花学报,2006,18(6)：347-351.
[95]Kumria R, Leelavathi S, Bhatnagar R, et al.Regeneration and genetic transformation of cotton: present status and future perspectives[J].Plant Tissue Cult,2003,13 (2):211-225.
[96]Leelavathi S, Sunnichan V, Kumria R, et al. A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.):Embryogenic calli as a source to generate large numbers of transgenic plants[J].Plant Cell Reports,2004,22 (7):465-470.
[97]Jin S, Zhang X, liang S, et al. Factors affecting transformation efficiency of embryogenic callus of Upland cotton (Gossypium hirsutum) with Agrobacterium tumefaciens[J].Plant cell,2005,81 (2): 229-237.
[98]Kim C, Chung J, Park S, et al. Agrobacterium tumefaciens-mediated transformation of Rosa hybrida using the green fluorescent protein (GFP) gene[J].Plant cell:tissue and organ culture,2004,78 (2): 107-111.
[99]Deyi X, Shuangxia J, Xiaoping G, et al. Somatic embryogenesis and plant regeneration in cotton cultivars from Yellow and Yangtze River planting areas [J].Acta Agronomica Sinica,2007,33: 1239-1254.
[100]严勇亮.棉花体细胞胚胎发生能力比较及IAP-P35基因遗传转化初步研究[D].乌鲁木齐：新疆 农业大学硕士学位论文,2009.
[101]杨国顺,谢丙炎,杨宇红,等.应用绿色荧光蛋白报告基因优化辣椒的遗传转化体系[J].园艺学报,2004,31(6)：737-742.
[102]陈大华,叶和春.绿色荧光蛋白基因在青蒿转基因芽中的表达[J].植物学报,1999,41(005)：490-493.
[103]贾小霞,张金文,王汉宁,等.抗真菌和抗除草剂基因多价植物表达载体构建及对烟草遗传转化的研究[J].草业学报,2009,18(1)：86.
[104]杜丽,曾晓慧,周索,等.GFP基因转化香樟胚性愈伤组织的研究[J].西北植物学报,2008,28(003)：465-469.
[105]李子银,胡会庆.农杆菌介导的植物遗传转化进展[J].生物工程进展,1998,18(1)：22-26.
[106]吴慎杰.陆地棉农杆菌介导遗传转化体系的优化与利用[D].南京：南京农业大学硕士学位论文,2006.