【作者】 余桂荣；

【导师】 尹钧； 郭天财；

【作者基本信息】 河南农业大学 ， 作物栽培学与耕作学， 2003， 硕士

【摘要】 本研究以小麦硫氧环蛋白反义基因(Anti-TrxS)为遗传转化的目的基因，进行了以下几个方面的研究：1、优良受体基因型的筛选与高效再生体系的优化；2、基因枪、花粉管通道及子房注射法转化目的基因；3、目的基因的检测。试验材料、方法及结果如下： 1．以19个栽培小麦品种(豫麦66、豫麦18-64、豫麦34、豫麦47、豫麦49、豫麦70、豫麦18、豫麦57、周麦16、89(117)、91(8)、偃高1号、郑麦9023、郑新991、郑新992、济麦2号、95519、00中13、国麦1号)的幼胚为试验材料，通过组织培养筛选适合基因枪法转化的优良受体基因型。研究结果表明：不同基因型对组织培养的反应有很大差异。豫麦70出愈率虽高(95.5％)，但愈伤组织分化特性却不好(分化率65.4％)；豫麦66分化率最高(85.5％)，而再生率却不高(21.7％)；国麦1号分化率只有39.1％，但分化出的绿芽点一半以上都能成苗；89(117)的出愈率、分化率和再生率都很高，分别为93.8％、83.3％、32.2％，是供试品种中最理想的基因型；豫麦18-64分化率虽偏低(66.9％)，但再生率达27.3％，远高于19个品种的平均水平。综合考虑各基因型在幼胚组织培养过程中的反应，89(117)和豫麦18-64是小麦基因枪法遗传转化的理想受体基因型。 2．在优化高效再生体系过程中，探讨了基因型、培养基及分化前的物理处理等因素对愈伤组织的诱导、分化和植株再生的影响。结果表明：不同基因型的幼胚在离体培养时，对胚龄、培养基和培养条件的要求不一样。在诱导愈伤组织时，不同基因型间虽有差异，但各基因型对附加不同激素组合的培养基的总体反应趋于一致；愈伤组织分化时，基因型与培养基的互作效应十分显著，基本培养基中附加激 余桂荣：小麦受体基因型与再生体系的优化及nti－TrxS基因的遗传转化 素的种类、浓度和配比对愈伤组织的诱导、分化具有显著的影响。愈 伤组织分化前的冷冻和暗培养处理能促进其快速分化出苗。本试验中 适于忧良基因型89门 人豫麦1864幼胚组织培养和植株再生的 方案为：剥出亘径为0．7叶．Zn的幼胚一盾片朝上接种于诱导培养 基IV（MS＋2，4－D 2 mg／L＋ABA 0．5 mg／L）上～暗培养 2～3周后， 挑选胚性较好的愈伤组织转移到继代培养基①mS＋2，4个 呷几＋ ABA 0．5 mg／L＋KT mg／L）上一每 15 天继代一次（继代 2一3次） 一置于4℃冰箱冷冻10大一挑选胚性愈伤组织转入分化培养基E或工 【为 MS＋ZT 4／NAA．5 mg／L；工为 MS＋6－BA 2／IAA 0．5 mg／L） 中进行光照培养一每 15天继代一次一将分化出的 2《 cm高的幼苗转 至生根培养基（l／2 MS＋琼脂8 g／L＋蔗糖80 g／L＋NAA 0．2 mg／L） 一向生根良好的三角瓶中注人清水，开盖练苗2亿天一转入花盆。 3．在基因转化与植株再生培养条件优化的基础上，以大面积推广 的白皮小麦豫麦1864的幼胚愈伤组织为受体材料，用基因枪法进行 了Anh1rxS基因和选择性标记3ar）基因的共转化。共轰击愈伤 组织 1200块，通过附加 3～5 mg／L Bialaphos的分化筛选培养基筛 选，筛选出 150块抗性愈伤组织，最后获得 32株再生植株，移栽成 活11 株。 4．以豫麦47、豫麦34、豫麦49、豫麦70、豫麦18、郑麦9023、 郑新99、郑新992、燥麦4号、00中13、济麦2号、95519和惬高 1号作为受体材料，用花粉管通道法进行了 Ant i｛rxS基因和 Bar基 因的共转化。共处理十三个小麦品种的2036朵小花，收获种子1616 粒，结实率为 79．4％，对转化所得 T。代种子大田点播成株行，T；代 共出苗 1424棵，出苗率为 88．l％。对 T；代苗按品种和处理组合的方 式进行抽样检测，抽样株系取 10个单株分别提取叶片基因组 DNA，然 2 河南农业大学硕士学位论文 后将单株叶片DNA等量混合，进行株系基因组DNA的PCR检测。检测 结果为31个株系中有16个株系呈阳性反应。 5．本研究过程中还尝试用子房注射法直接向花期小麦子房中注 入载有目的基因的外源质粒DNA。试验材料为豫麦49、豫麦70、豫 麦18、郑新991、95519五个小麦品种。共转化小花1206朵，收获 种子 89粒，结实率为 7．4％，T；代共出苗 41棵，出苗率为 46．l％。 抽取郑新99；代株系Q2棵幼苗的叶片基因组DNA混合样）进行 PCR检测，电泳结果呈阳性反应。更多还原

【Abstract】 The objective for this study was to transfer Anti-TrxS into wheat cultivars. Three aspects was studied in this paper. Firstly , selecting the optimal genotypes as receptors for wheat genetic transformation and optimize the culture system of immature embryo; Secondly, transferring Anti-TrxS into wheat by methods of microprojectile bombardment, pull en tube pathway and ovary injection;Thirdly, analyzing Anti-TrxS Gene of the transformed plants by PCR. The main results were as follows:1. In order to select the optimal wheat genotype for immature embryo culture and gene transformation. 19 wheat genotypes were studied. The result showed that the difference among the genotypes was significant. Yumai70 had high rate of callus induction (95.5%), but the frequency of green primordial differentiation was only 65.4%; Yumai66 had the highest rate of green primordial differentiation, but the frequency of plant regeneratation was only 21.7%;Guomai No.l had the lowest frequency of green primordial differentiation(39.1%), but half of the green primordials could be differentiated into plantlets; 89 (117) had high frequency of callus induction (93.8%), green primordial differentiation’s.3%) and plant regeneratation(32.2%) respectively. So it was the best wheat genotype for genetic transformation. Yumai 18-64 also had the higher frequency of plant regeneratation(2 7.3 %).2. The factors influencing culture ability of immature embryo,such as genotype, medium and physical treatment were studied in the process of the immature embryo culture.The result showed that immature embryos of different genotypes required different embryo age, medium and culture condition. Media with different ratios of exogenous hormones effect the differntiation ability of immature embryossignificantly. In the inducation culture,there was difference among different genotypes,but they had a same trend. There was significant interaction between genotype and medium in the differentiation culture. The treatments of desiccation, refrigeration before the differentiation culture can significantly affect the plant regeneration of the calli.The optimal culture system for immature embryo from 89(117), Yumai18-64 was 0.7-1.2 mm(dimeter)immature embryo in the size-on the inducation medium IV (MS + 2,4-D 2 mg/L + ABA 0.5 mg/L) and dark culture for 20 days -on subculture medium?(MS + 2,4-D 1 mg/L + ABA 0.5 mg/L + KT 1 mg/L) for 15 days -refrigeration for 10 days-on the differentiation medium E or I(E:MS+ZT4/NAA/1.5mg/L; LMS+6-BA2/IAA 0.5mg/L) for 15 days-on rootage medium-translpant into pots.3. Based on the optimization of genotype and culture system, the Anti-TrxS and bar genes were co-transferred to the immature embryos (calli) from Yumail8-64 as receptor by bombardment. 3~5 mg/L Bialaphos was added into the medium to select resistant callus during callus induction and regeneration culture. 32 regenerated plantlets were obtained and 11 of them grown normally.4. Anti-TrxS. and Bar genes were co-transferred to 2036 flowers of receptors, Yumai18, Yumai34,Yumai47, Yumai49,Yumai70,Zhengmai9023,OOzhong13, Jimai No.2, YangaoNo.1, 95519, Zhengxin99K Zhengxin992 and Luomai No.4 through pullen tube pathway. 1616 seeds from transferred receptors with a percentage of 79.4 % and 1424 plants from1616 seeds with a percentage of 88.1% were obtained. Leaf genome DNA from T1 plant of different genotypes and treataments were analyzed by PCR and 16 positive lines were obtained among 31 tested lines.5. Two genes were also co-transferred to 1206 flowers of Yumail8,Yumai49 Yumai70,Zhengxin991, 95519 by methods of ovary injection. 89 seeds (7.4% of the flowers) and 41 plants (46.1% of seeds)were achieved. Leaf genome DNA of Zhengxin991 TI plants strain was identified by PCR analysis.更多还原