【作者】 刘香利；

【导师】 郭蔼光；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 小麦是世界上栽培面积最大的重要粮食作物。然而,小麦却是最后一个获得转化成功的重要禾谷类作物。主要原因就是目前常用的转化方法无论是基因枪法还是农杆菌介导法都依赖于组织培养技术,而小麦不同外植体组织培养受基因型影响很大,使其遗传转化集中在少数几种组织培养再生能力较高的品种上,而生产上大面积栽培品种由于再生频率低转化较困难,使得小麦的转基因工作进展缓慢,难以应用于育种实践。随着食品加工业的发展和人民生活水平的提高,对优质面包小麦的需求量日益增加。大量研究表明,小麦高分子量麦谷蛋白亚基(HMW-GS)组成与小麦加工品质密切相关。因此,利用优质高分子量麦谷蛋白亚基基因改良小麦品质具有重要理论及实践意义。本研究在克隆了小麦优质HMW-GS 1Bx14基因的基础上对生产上大面积种植的小麦品种组织培养特性进行研究,并利用基因枪和农杆菌转化法对1Bx14基因进行转化,同时对花粉管通道法转化法进行了初步探索,取得以下结果:1.对生产上大面积种植的不含1Bx14基因的小麦品种幼穗、幼胚和成熟胚组织培养特性进行研究,分析了不同小麦品种、不同外植体的出愈率及其愈伤组织的分化和再生潜力及其培养条件。结果表明,小麦不同品种和不同外植体愈伤组织诱导率均较高,都在90%以上,且不同品种间差异不大;而愈伤组织分化和植株再生率品种间和不同外植体间均存在很大差异;愈伤组织的诱导、分化和植株的再生是相互独立的,诱导率高并不表明分化率高,分化率高的品种再生率不一定高。同一品种不同外植体,幼胚愈伤组织分化和植株再生率最高,幼穗次之,成熟胚培养效果最差。不同小麦品种幼穗愈伤组织分化率依次为:绵阳19>洛阳8716>小偃107>陕354>小偃22>陕150>郑麦9023>西农88;幼胚分化能力依次为:绵阳19>小偃107>小偃22>郑麦9023>洛阳8716>陕354。成熟胚分化能力依次为:陕354>绵阳19>洛阳8716>小偃107>小偃22>郑麦9023。总体来说,绵阳19和小偃107幼胚、绵阳19和洛阳8716幼穗,以及陕354和绵阳19的成熟胚愈伤组织分化率相对较高,可作为遗传转化的受体材料。其中,绵阳19幼胚愈伤组织再生率最高,且多数为丛生苗,是转化最理想的材料。2.以绵阳19幼胚愈伤组织为材料,利用仅含有受控于1Dx2胚乳特异性启动子的1Bx14基因及其3-UTR的线性DNA表达框转化小麦幼胚愈伤组织。共转化小麦幼胚愈伤组织2480块,利用基因池法进行PCR筛选,从1219个转化再生植株中共检测出7株阳性单株,平均转化效率0.28%。7个转基因植株中有4个生长正常,3个植株出现表型变异(A1-14,B2-8和D5-10),表现为植株矮小,仅1～2个分蘖,且分蘖不能成穗,主茎成穗且穗较小,其中B2-8和D5-10未能结实,A1-14开花结实较晚,仅收获3粒种子。对5个正常结实的阳性植株所得的82个转化T1代种子用半粒法进行SDS-PAGE分析,L2、L4和L5三个后代能检测到14亚基的表达,但表达量相对内源高分子量麦谷蛋白亚基较低;转基因植株A1-14的三株T1代植株在大田均表现为雄性不育。初步建立了借助于PCR筛选的无标记转化体系。3.构建了胚乳特异性启动子驱动的HMW-GS 1Bx14基因植物表达载体,确立了绵阳19幼胚愈伤组织最适潮霉素筛选浓度50 mg/L,并利用基因枪法进行载体转化。共转化绵阳19幼胚愈伤组织652块,转化后的愈伤组织进行潮霉素筛选,共有11块愈伤组织分化出小植株,移栽后有8株存活,PCR和PCR-Southern结果显示7株为转化阳性植株,Southern杂交结果显示有6株转基因稳定整合,转化效率0.92%。SDS-PAGE结果显示,有三个株系后代中检测到HMW-GS 1Bx14基因表达,但其他亚基都受到了不同程度的抑制。4.利用GUS瞬时表达对农杆菌转化体系进行优化,结果表明洛阳8716幼胚愈伤组织对LBA4404的敏感性高于绵阳19。EHA105对于两个品种幼胚愈伤组织侵染率都较LBA4404高。对于绵阳19幼胚愈伤组织,EHA105侵染的最佳参数为OD600 0.8×30 min,LBA4404为OD600 1.0×30 min;对于洛阳8716幼胚愈伤组织,EHA105侵染的最佳参数为OD600 0.8×30 min,LBA4404为OD600 0.8×60 min。利用LBA4404侵染绵阳19幼胚愈伤组织共483块,经潮霉素筛选和PCR分析后得到2株阳性植株;侵染洛阳8716愈伤组织321块,筛选后仅得到1株阳性植株。利用EHA105转化小麦绵阳19幼胚愈伤组织452块,筛选后得到5株阳性植株,侵染洛阳8716愈伤组织315块,得到2株阳性植株,所有处理平均转化率0.61%。5.以5个不含高分子量麦谷蛋白14亚基的小麦栽培品种为材料,对花粉管通道法转化条件进行了初步摸索。对转化后代利用基因池法进行PCR分析,结果表明,不同小麦品种不同处理间转化率存在很大差异,平均转化率0.56%。其中陕354处理2的转化率最高,为1.01%,其次为陕354处理4为0.93%。陕893的处理1、处理3和小偃107的处理1转化率也均较高,在0.8%以上。初步证明使用花粉管通道法对小麦进行遗传转化是可行的。更多还原

【Abstract】 Wheat is one of the most important food crop which is planted widely in the world. However, wheat is the last one of the gramineous crop which was transformed successfully. The main reason is that the transformation mediated by Agrobacterium tumefaciens or microprojectile bombardment depend on tissue culture and regeneration, which is influenced greatly by genotype. Only a few highly regenerated varieties can be transformed, while a large quantity of elite cultivars can not be transformed successfully due to their poor regeneration property. This result in the transformation of wheat develops slowly and can hardly be applied in breeding practice. The development of foodstuffs processing and improvement of living standard have created a great demand for high quality bread-making wheat. The high molecular weight glutenin subunit (HMW-GS) are closely associated with the bread-making quality of wheat flour. Therefore, It is theoretically and practically significant to use genetic transformation of high quality HMW-GS gene to improve wheat quality.This investigation, based on the clone of HMW-GS 1Bx14 gene, the plant regeneration system of elite wheat cultivars was studied, and the immature embryo was transformed by Agrobacterium tumefaciens and microprojectile bombardment with the high quality HMW-GS gene 1Bx14. The method of pollen tube pathway was also explored. The major results are as follows:1. The callus induction and plant regeneration of immature embryo, immature inflorescences and mature embryo from different common wheat cultivars was investigated. The result showed that the frequency of callus induction was higher than 90 percent and there was no significant difference between explants and varieties. While the frequency of callus differentiation and plantlet regeneration of different wheat Varieties and explants are quite different. Callus induction, differentiation and regeneration were independent, and there was no significant correlation among them. Generally, the callus differentiation and plantlet regeneration frequency of immature embryo was highest, immature inflorescence take second place, and mature embryo is lowest. The frequency of immature inflorescence callus differentiation rank downward in the order Mianyang 19, Luoyang 8716, Xiaoyan107, Shaan354, Xiaoyan22, Shaan150, Zhengmai 9023, Xinong 88. The frequency of immature embryo callus differentiation, from high to low, was Mianyang 19, Xiaoyan107, Xiaoyan22, Zhengmai 9023, Luoyang 8716, Shaan354. The frequency of mature embryo callus differentiation was Shaan354 > Mianyang 19 > Luoyang 8716 > Xiaoyan107 > Xiaoyan22 > Zhengmai 9023. Most of the differentiated callus of Mianyang19 formed multiple shoot clumps and the frequency of regeneration was highest among all the varieties, therefore the immature embryo of Mianyang 19 can be the most ideal explants for transformation.2. In order to improve bread-making quality of flour and produce transgenic plants free of selectable markers, a linear DNA construct consisting of a minimal expression cassette with the HMW-GS 1Bx14 gene was transformed into wheat cultivar Mianyang19 by microprojectile bombardment. The transformants were selected by PCR instead of markers gene. 2480 callus of Mianyang19 was transformed and seven transgenic plants were identified from a total of 1,219 transformants, yielding a transformation frequency of 0.28%. Four of the seven transformants have the normal phenotype, and the other three(A1-14, B2-8 and D5-10)demonstrate phenotypic variation among which two transformants (B2-8 and D5-10) could not form seeds and A1-14 had only 3 seeds. An SDS-PAGE analysis of 82 T1 seeds confirmed that the 1Bx14 gene was expressed in the progenies of three transgenic T1 seeds (L2, L4 and L5), while the expression was weaker than other endogenous subunits. The three T1 progenies of A1-14 were male sterility. it is feasible to obtain marker-free transformants using the linear expression cassette transformation approach coupled with PCR selection.3.The plant expression vector of HMW-GS 1Bx14 gene was constructed, and a total of 652 callus of Mianyang 19 immature embryo was transformed by microprojectile bombardment. After selected with hygromycin, 11 callus was differentiated to plantlet and 8 of which survived in greenhouse. The result of PCR and PCR-southern showed that 1Bx14 have been transformed into seven plantlet and the frequency of transformation was 1.07%. Three transgenic plants demonstrated the expression of transgene and the expression of other subunits was suppressed in varying degrees.4. The genetic transformation systems of wheat Mediated by Agrobacterium was investigated. The result showed that Luoyang 8716 was more sensitive to Agrobacterium strain LBA4404 than Mianyang 19. For the two varieties, the infection frequency of Agrobacterium strain EHA105 was higher than LBA4404. The optimal procedure to Mianyang was OD600 0.8×30 min and OD600 1.0×30 min for strain EHA105 and LBA4404, respectively. To Luoyang 8716, the Optimum procedure was OD600 0.8×30 min and OD600 0.8×60 min, respectively. Selected by hygromycin and analyzed by PCR, two positive plant was gained from 483 callus of Miangyang 19, and one was got from 321 callus of Luoyang 8716 using Agrobacterium strain LBA4404. Using Agrobacterium strain EHA105,five positive plant was regenerated from 452 callus of Miangyang 19, and two from 315 callus of Luoyang 8716.5. The HMW-GS 1Bx14 gene was transformed into five wheat varieties by pollen tube pathway, and transformants were identified by PCR. The result showed that the differences between varieties and treatments were significant, and the average frequency of transformation was 0.56 %. The transformation frequency of Shaan354 by treatment 2 was the highest (1.01%), follow by treatment 4 (0.93%). The frequency of Shaan 893 by treatment 1, treatment 3 and Xiaoyan 107 by treatment 1 was over 0.8%. Therefore it is a potential way to transform wheat by pollen tube pathway.更多还原