利用转基因聚合创造高淀粉、高直链淀粉玉米新种质

发布时间：2018-02-01 04:10

本文关键词： 玉米 AGPase GBSSI SBEIIRNAi ae突变体转基因高淀粉高直链淀粉　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：玉米是世界第一大作物,其籽粒可作为粮食、饲料和工业原料,淀粉含量和品质是决定玉米经济价值的重要因子。高淀粉玉米工业价值高,可提高单位面积上的玉米产值;高直链淀粉玉米可应用于产生高直链淀粉,后者是食品包装薄膜、可降解塑料等产品的原料。因此,通过传统育种或转基因的方式获得高淀粉与高直链淀粉玉米的研究具有十分重要的意义。本实验室前期研究表明:将Sh2r6hs基因和Bt2基因同时转入不同的玉米自交系中,与对照相比转基因材料的总淀粉含量、直链淀粉含量及百粒重都显著增加。过量表达ZmWx基因同样能够提高转基因过表达玉米的淀粉含量及百粒重。通过RNAi技术同时抑制玉米骨干自交系昌7-2中SBEIIb、SBEIIa基因表达,测得直链淀粉含量由对照的28%增加到50%以上,但总淀粉含量降低,由对照的66%降到60%以下。转基因玉米胚乳淀粉粒及传递细胞的观察为研究以上转基因玉米淀粉含量和籽粒大小变化的原因,通过常规石蜡切片法制片,PAS反应(Periodic acid Schiff reaction)显色或甲苯胺蓝-O染色,光学显微镜下观察了淀粉粒、传递细胞和淀粉含量的变化。利用扫描电镜观察了以上转基因材料粉质胚乳的中心处的淀粉粒形态、大小,并测量了淀粉粒直径。通过石蜡切片观察得出,Sh2r6hsBt2基因过表达玉米、ZmWx基因过表达玉米胚乳中的淀粉粒要明显多于对照,sBEIIRNAi转基因玉米胚乳中的淀粉粒要明显少于对照;而传递细胞未发生明显变化。通过扫描电镜观察得出,Sh2r6hsBt2过表达材料、ZmWx过表达材料的淀粉粒直径的分布范围和平均值均大于受体自交系的;与对照相比,以昌7-2为受体的转SBEIIRNAi结构的玉米淀粉粒表面出现不规则的塌陷或凹陷;且从淀粉粒直径分布来看,转SBEIIRNAi结构的株系明显降低了直径在8-13μm的淀粉粒比例。因此,转Sh2r6hsBt2基因玉米、转ZmWx基因玉米、转SBEIIRNAi结构玉米的淀粉含量及籽粒大小的改变,与淀粉粒的数量及大小变化相对应,但是与传递细胞数量的变化没有明显的关系,表明Sh2r6hsBt2基因、ZmWx基因过表达主要使AGPase、GBSSI酶活性提高,合成淀粉的效率提高;而SBEIIRNAi结构使SBEII酶活性降低,影响淀粉的合成。GBSSI过表达玉米与转突变型AGPase玉米的杂交对直链淀粉含量的影响郑58、昌7-2是两个优良自交系,二者具有很好的配合力,郑单958即为以郑58为母本、昌7-2为父本组配的杂交种。本实验将编码GBSSI的ZmWx基因过表达玉米与编码突变型AGPase的Sh2r6hsBt2基因过表达玉米进行杂交,且突变型AGPase转基因玉米来自昌7-2,GBSSI转基因玉米来自郑58,以期培育出总淀粉含量和直链淀粉高的转基因郑单958,了解其在农业生产上的应用价值。实验对转基因聚合玉米进行了总淀粉含量、直链淀粉含量的测定,并对籽粒性状、果穗性状、百粒重、产量进行了分析。与对照杂交材料相比,转基因聚合材料的淀粉、直链淀粉含量提高,株高、穗位高没有明显变化,但穗长、行粒数、百粒重、单株产量增加。以郑58为母本的聚合材料,单株产量由非转基因郑单958的169.4 ± 6.23g增加到200.0 ± 4.56g。因此,转基因杂交种与对照杂交种相比既提高了淀粉含量,又增加了直链淀粉含量,且转基因杂交种与对照杂交种相比单株产量显著增加,百粒重增加和果穗籽粒数增多共同引起单株产量的增加。将该种子应用于农业生产,可明显提高玉米产量,增加农民的收益。ae突变体材料与不同转基因材料的杂交分析为了能够培育出高直链淀粉玉米新种质,本实验将ae突变体玉米分别与转SBEIIRNAi结构玉米、突变型AGPase过表达玉米、ZmWx基因过表达玉米杂交。测定了转基因聚合种子淀粉、直链淀粉的含量。淀粉含量测定结果表明,昌7-2背景下ae突变体的直链淀粉含量达到56.9%,郑58背景下ae突变体的直链淀粉含量达到55.1%,ae突变体与转SBEIIRNAi结构聚合玉米的直链淀粉含量除两个较特殊的外最高达33.9%。ae突变体与ZmWx基因过表达聚合玉米的直链淀粉含量最高达37.1%;与转突变型AGPase基因聚合玉米的直链淀粉含量也明显低于亲本ae突变体。且以ae突变体为母本的聚合材料直链淀粉含量要高于以转基因玉米为母本的,表现出母本效应。由以上结果得出,转基因聚合种子直链淀粉含量没有提高,反而明显低于亲本ae突变体,可能是转基因聚合种子的胚乳中,存在野生型SBEⅡb基因导致的。由于时间关系,尚未得到聚合ae基因的纯合材料。转AGPase基因和转突变型AGPase基因株系的杂交聚合本实验将Sh2Bt2过表达玉米与Sh2r6hsBt2过表达玉米杂交,增加AGPase大亚基的杂合性,尝试能否进一步提高玉米淀粉含量。对转基因聚合玉米的淀粉含量、百粒重进行了测定,并且对果穗、籽粒进行了表型分析。实验结果表明,以Sh2Bt2基因过表达玉米为母本的聚合材料淀粉含量达到76.7%、78.6%,百粒重为 24.9±1.74g、26.2±0.65g;以Sh2r6hsBt 基因过表达玉米为母本的聚合材料淀粉含量达到78.0%、81.1%,百粒重为26.7 ±0.51g、27.9±0.60g;均高于亲本。由以上结果可知,Sh2Bt2基因过表达植株与Sh2r6hsBt2基因过表达植株杂交,产生的种子具有增加的淀粉含量和百粒重,与亲本相比差异达到显著水平。通过籽粒、果穗的表型分析,转基因聚合材料的籽粒变大、果穗变大。综上所述,本论文通过观察不同转基因玉米淀粉粒、传递细胞和淀粉含量的变化,得出淀粉含量的变化与淀粉粒的数量及大小变化相对应,但与传递细胞没有明显的关系。将不同自交系的GBSSI过表达材料与突变型AGPase过表达材料进行杂交,培育出总淀粉含量和直链淀粉高的转基因玉米材料。ae突变体玉米与不同转基因玉米的杂交,虽未得到聚合ae基因的纯合材料,聚合种子的直链淀粉含量低于亲本,但这为高直链淀粉玉米新种质的创制提供了思路。转野生型AGPase基因和转突变型AGPase基因株系的杂交,聚合种子与亲本相比具有增加的淀粉含量和百粒重。以上研究,为转基因高淀粉、高直链淀粉玉米的研究提供了策略和方法,选育的高淀粉、高直链淀粉材料具有一定的应用前景。
[Abstract]:Corn is the world's largest crop, the grain can be used as food, feed and industrial raw materials, the content and quality of starch is an important factor in determining the economic value of corn. High starch corn industry value is high, can improve the unit area of corn production; high amylose maize can be applied to produce high amylose starch, which is food packaging film, degradable plastic products such as raw materials. Therefore, high starch and high amylose corn straight research has very important significance to get through traditional breeding or transgenic approaches. Our previous studies showed that the Sh2r6hs gene and the Bt2 gene into different maize inbred lines, compared with the total starch content transgenic materials, amylose content and 100 grain weight increased significantly. Overexpression of ZmWx can improve the expression of transgenic maize starch content and 100 grain weight by RNAi. At the same time, inhibition technology of maize inbred line 7-2 Chang SBEIIb, SBEIIa gene expression, measured amylose content by 28% of the control increased to more than 50%, but the total starch content decreased from 66% in the control below 60%. Due to the above research on genetically modified maize starch content and grain size of the changes of genetically modified corn starch particle and transfer cells, by conventional paraffin method, the reaction of PAS (Periodic acid Schiff reaction) color or toluidine blue -O staining under optical microscope the changes of starch grains, transfer cells and starch content were observed. At the center of the above transgenic material powder endosperm starch grain morphology by scanning electron microscopy, size and the starch grain diameter was measured. Paraffin sections through the observation that the over expression of Sh2r6hsBt2 gene of maize ZmWx gene over expression of starch grains in maize endosperm were significantly more than the control SBEIIRNAi, the starch grains in the endosperm of transgenic corn is significantly less than the control; while the transfer cells did not change significantly. By using scanning electron microscope showed that the overexpression of Sh2r6hsBt2, ZmWx over expression of starch granule size distribution range of the materials and the average values were greater than the recipient inbred line; compared with the control, in order to Chang 7-2. SBEIIRNAi the structure of the receptor of corn starch granules appear on the surface of irregular subsidence or depression; and from the starch granule size distribution, structure of transgenic SBEIIRNAi lines was significantly reduced starch in 8-13 m particle diameter ratio. Therefore, Sh2r6hsBt2 transgenic maize, ZmWx transgenic maize, transgenic SBEIIRNAi maize starch content and grain structure size changes, corresponding changes in the number and size of starch grains, but no significant relationship with the change of the number of transfer cells, suggesting that the Sh2r6hsBt2 gene, ZmWx gene expression mainly The AGPase, increased GBSSI activity, improve the efficiency of the synthesis of starch; and the SBEIIRNAi structure of the enzymatic activity of SBEII decreased, affecting the synthesis of.GBSSI starch and the effect of overexpression of mutant AGPase Transgenic Maize Hybrid Maize on amylose content of Zheng 58, Chang 7-2 is the two inbred lines, two with force well, Zhengdan 958 is to Zheng 58 as female parent and hybrid male parent Chang 7-2 group. The ZmWx gene encoding the GBSSI expression of Sh2r6hsBt2 gene in maize and encoding mutant AGPase overexpression of Maize Hybrid and mutant AGPase transgenic maize from Chang 7-2, GBSSI transgenic maize from Zheng 58, in order to cultivate a total starch content and amylose of transgenic Zhengdan 958, understand its application value in agricultural production. Experiment of total starch content of transgenic maize determined polymerization, amylose content, and seed Grain traits, ear traits, 100 grain weight and yield were analyzed. Compared with the control of hybrid materials, polymeric materials genetically modified starch, amylose content increased, plant height, ear height did not change significantly, but the panicle length, grain number, grain weight, yield per plant increased by 58 for the polymerization of Zheng. The parent material, the yield from non transgenic Zhengdan 958 increased to 169.4 + 6.23g 200 + 4.56g. therefore, transgenic hybrid and hybrid control compared to both improved and increased starch content, amylose content, and transgenic hybrid and hybrid control compared to the yield per plant increased significantly, 100 grain weight was increased and the number of ear kernels due to the increased yield per plant. The seeds used in agricultural production, can significantly increase the yield of maize, increase farmer's income.Ae mutant materials and different transgenic materials hybridization analysis in order to be able to produce high straight The new germplasm of amylose maize, the maize AE mutant and transgenic SBEIIRNAi maize respectively, the overexpression of AGPase gene in maize, overexpression of ZmWx gene. The transgenic maize hybrid seed polymerization determination of starch and amylose content were measured. The results showed that the starch content, under the background of Chang 7-2 AE mutant amylose content reached 56.9%, Zheng 58 under the background of the AE mutant amylose content reached 55.1%, AE mutant and transgenic SBEIIRNAi structure polymerization of corn amylose content in addition to the two special outside up to 33.9%.ae mutant and ZmWx gene over expression of amylose content in polymerization of corn up to 37.1%; the amylose content of maize and turn the mutant AGPase gene pyramiding is lower than that of the parental AE mutants. And as the female parent polymer amylose content was higher than that in female transgenic maize with AE mutants showed a parent This effect is obtained by the above results, the transgenic seed polymerization of amylose content was not increased, but significantly lower than the parental AE mutants may be genetically modified seed endosperm polymerization in the presence of wild-type B gene leads to SBE II. Because of the time, has not yet been polymerized ae gene homozygous transgenic AGPase and hybrid materials. Transgenic mutant AGPase strains of the polymerization of this experiment will be over expression of Sh2Bt2 and overexpression of Sh2r6hsBt2 in Maize Hybrid Maize, increase of AGPase large subunit of heterozygosity, try can improve the content of corn starch. The starch content of transgenic corn polymerization, 100 grain weight was measured, and the ear, grain of phenotype analysis. The experimental results show that Sh2Bt2 overexpression of corn starch content in female polymeric materials reached 76.7%, 78.6%, 100 grain weight was 24.9 + 1.74g, 26.2 + 0.65g; Sh2r6hsBt gene over expression As for the corn starch content of female polymeric materials reached 78%, 81.1%, 100 grain weight was 26.7 + 0.51g, 27.9 + 0.60g; both were higher. The results above indicated that over expression of Sh2Bt2 gene and Sh2r6hsBt2 gene expression of plant hybrids, produce seeds with starch content increased and 100 grain weight, and compared to the parents there were significant differences. Through the analysis of ear of grain, the grain of transgenic phenotype, polymeric material becomes larger, the ear became large. To sum up, through the observation of different genetically modified corn starch grains, the change of transfer cells and starch content, the size and number of changes in the starch grains that starch content should be relative, but no obvious relationship between the transfer cells. Different inbred lines GBSSI overexpression of mutated AGPase expression materials and materials produced transgenic maize hybrids, total starch content and amylose high .ae mutants of Maize with different transgenic maize hybrids, has not yet been homozygous polymerization of AE gene, amylose content were lower than that of the seed polymerization, but the high amylose maize germplasm creation directly provides ideas. Hybridization of wild type AGPase gene and AGPase gene mutant strain. And compared with the parent seed polymerization of starch content increased and 100 grain weight. The above research, transgenic high starch, provides strategies and methods for the study of high amylose maize, high starch breeding, high amylose starch material has a certain application prospect.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S513

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