發(fā)布時間：2018-02-01 04:10

  本文關(guān)鍵詞： 玉米 AGPase GBSSI SBEIIRNAi ae突變體 轉(zhuǎn)基因 高淀粉 高直鏈淀粉　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：玉米是世界第一大作物,其籽�？勺鳛榧Z食、飼料和工業(yè)原料,淀粉含量和品質(zhì)是決定玉米經(jīng)濟(jì)價值的重要因子。高淀粉玉米工業(yè)價值高,可提高單位面積上的玉米產(chǎn)值;高直鏈淀粉玉米可應(yīng)用于產(chǎn)生高直鏈淀粉,后者是食品包裝薄膜、可降解塑料等產(chǎn)品的原料。因此,通過傳統(tǒng)育種或轉(zhuǎn)基因的方式獲得高淀粉與高直鏈淀粉玉米的研究具有十分重要的意義。本實驗室前期研究表明:將Sh2r6hs基因和Bt2基因同時轉(zhuǎn)入不同的玉米自交系中,與對照相比轉(zhuǎn)基因材料的總淀粉含量、直鏈淀粉含量及百粒重都顯著增加。過量表達(dá)ZmWx基因同樣能夠提高轉(zhuǎn)基因過表達(dá)玉米的淀粉含量及百粒重。通過RNAi技術(shù)同時抑制玉米骨干自交系昌7-2中SBEIIb、SBEIIa基因表達(dá),測得直鏈淀粉含量由對照的28%增加到50%以上,但總淀粉含量降低,由對照的66%降到60%以下。轉(zhuǎn)基因玉米胚乳淀粉粒及傳遞細(xì)胞的觀察為研究以上轉(zhuǎn)基因玉米淀粉含量和籽粒大小變化的原因,通過常規(guī)石蠟切片法制片,PAS反應(yīng)(Periodic acid Schiff reaction)顯色或甲苯胺藍(lán)-O染色,光學(xué)顯微鏡下觀察了淀粉粒、傳遞細(xì)胞和淀粉含量的變化。利用掃描電鏡觀察了以上轉(zhuǎn)基因材料粉質(zhì)胚乳的中心處的淀粉粒形態(tài)、大小,并測量了淀粉粒直徑。通過石蠟切片觀察得出,Sh2r6hsBt2基因過表達(dá)玉米、ZmWx基因過表達(dá)玉米胚乳中的淀粉粒要明顯多于對照,sBEIIRNAi轉(zhuǎn)基因玉米胚乳中的淀粉粒要明顯少于對照;而傳遞細(xì)胞未發(fā)生明顯變化。通過掃描電鏡觀察得出,Sh2r6hsBt2過表達(dá)材料、ZmWx過表達(dá)材料的淀粉粒直徑的分布范圍和平均值均大于受體自交系的;與對照相比,以昌7-2為受體的轉(zhuǎn)SBEIIRNAi結(jié)構(gòu)的玉米淀粉粒表面出現(xiàn)不規(guī)則的塌陷或凹陷;且從淀粉粒直徑分布來看,轉(zhuǎn)SBEIIRNAi結(jié)構(gòu)的株系明顯降低了直徑在8-13μm的淀粉粒比例。因此,轉(zhuǎn)Sh2r6hsBt2基因玉米、轉(zhuǎn)ZmWx基因玉米、轉(zhuǎn)SBEIIRNAi結(jié)構(gòu)玉米的淀粉含量及籽粒大小的改變,與淀粉粒的數(shù)量及大小變化相對應(yīng),但是與傳遞細(xì)胞數(shù)量的變化沒有明顯的關(guān)系,表明Sh2r6hsBt2基因、ZmWx基因過表達(dá)主要使AGPase、GBSSI酶活性提高,合成淀粉的效率提高;而SBEIIRNAi結(jié)構(gòu)使SBEII酶活性降低,影響淀粉的合成。GBSSI過表達(dá)玉米與轉(zhuǎn)突變型AGPase玉米的雜交對直鏈淀粉含量的影響鄭58、昌7-2是兩個優(yōu)良自交系,二者具有很好的配合力,鄭單958即為以鄭58為母本、昌7-2為父本組配的雜交種。本實驗將編碼GBSSI的ZmWx基因過表達(dá)玉米與編碼突變型AGPase的Sh2r6hsBt2基因過表達(dá)玉米進(jìn)行雜交,且突變型AGPase轉(zhuǎn)基因玉米來自昌7-2,GBSSI轉(zhuǎn)基因玉米來自鄭58,以期培育出總淀粉含量和直鏈淀粉高的轉(zhuǎn)基因鄭單958,了解其在農(nóng)業(yè)生產(chǎn)上的應(yīng)用價值。實驗對轉(zhuǎn)基因聚合玉米進(jìn)行了總淀粉含量、直鏈淀粉含量的測定,并對籽粒性狀、果穗性狀、百粒重、產(chǎn)量進(jìn)行了分析。與對照雜交材料相比,轉(zhuǎn)基因聚合材料的淀粉、直鏈淀粉含量提高,株高、穗位高沒有明顯變化,但穗長、行粒數(shù)、百粒重、單株產(chǎn)量增加。以鄭58為母本的聚合材料,單株產(chǎn)量由非轉(zhuǎn)基因鄭單958的169.4 ± 6.23g增加到200.0 ± 4.56g。因此,轉(zhuǎn)基因雜交種與對照雜交種相比既提高了淀粉含量,又增加了直鏈淀粉含量,且轉(zhuǎn)基因雜交種與對照雜交種相比單株產(chǎn)量顯著增加,百粒重增加和果穗籽粒數(shù)增多共同引起單株產(chǎn)量的增加。將該種子應(yīng)用于農(nóng)業(yè)生產(chǎn),可明顯提高玉米產(chǎn)量,增加農(nóng)民的收益。ae突變體材料與不同轉(zhuǎn)基因材料的雜交分析為了能夠培育出高直鏈淀粉玉米新種質(zhì),本實驗將ae突變體玉米分別與轉(zhuǎn)SBEIIRNAi結(jié)構(gòu)玉米、突變型AGPase過表達(dá)玉米、ZmWx基因過表達(dá)玉米雜交。測定了轉(zhuǎn)基因聚合種子淀粉、直鏈淀粉的含量。淀粉含量測定結(jié)果表明,昌7-2背景下ae突變體的直鏈淀粉含量達(dá)到56.9%,鄭58背景下ae突變體的直鏈淀粉含量達(dá)到55.1%,ae突變體與轉(zhuǎn)SBEIIRNAi結(jié)構(gòu)聚合玉米的直鏈淀粉含量除兩個較特殊的外最高達(dá)33.9%。ae突變體與ZmWx基因過表達(dá)聚合玉米的直鏈淀粉含量最高達(dá)37.1%;與轉(zhuǎn)突變型AGPase基因聚合玉米的直鏈淀粉含量也明顯低于親本ae突變體。且以ae突變體為母本的聚合材料直鏈淀粉含量要高于以轉(zhuǎn)基因玉米為母本的,表現(xiàn)出母本效應(yīng)。由以上結(jié)果得出,轉(zhuǎn)基因聚合種子直鏈淀粉含量沒有提高,反而明顯低于親本ae突變體,可能是轉(zhuǎn)基因聚合種子的胚乳中,存在野生型SBEⅡb基因?qū)е碌�。由于時間關(guān)系,尚未得到聚合ae基因的純合材料。轉(zhuǎn)AGPase基因和轉(zhuǎn)突變型AGPase基因株系的雜交聚合本實驗將Sh2Bt2過表達(dá)玉米與Sh2r6hsBt2過表達(dá)玉米雜交,增加AGPase大亞基的雜合性,嘗試能否進(jìn)一步提高玉米淀粉含量。對轉(zhuǎn)基因聚合玉米的淀粉含量、百粒重進(jìn)行了測定,并且對果穗、籽粒進(jìn)行了表型分析。實驗結(jié)果表明,以Sh2Bt2基因過表達(dá)玉米為母本的聚合材料淀粉含量達(dá)到76.7%、78.6%,百粒重為 24.9±1.74g、26.2±0.65g;以Sh2r6hsBt 基因過表達(dá)玉米為母本的聚合材料淀粉含量達(dá)到78.0%、81.1%,百粒重為26.7 ±0.51g、27.9±0.60g;均高于親本。由以上結(jié)果可知,Sh2Bt2基因過表達(dá)植株與Sh2r6hsBt2基因過表達(dá)植株雜交,產(chǎn)生的種子具有增加的淀粉含量和百粒重,與親本相比差異達(dá)到顯著水平。通過籽粒、果穗的表型分析,轉(zhuǎn)基因聚合材料的籽粒變大、果穗變大。綜上所述,本論文通過觀察不同轉(zhuǎn)基因玉米淀粉粒、傳遞細(xì)胞和淀粉含量的變化,得出淀粉含量的變化與淀粉粒的數(shù)量及大小變化相對應(yīng),但與傳遞細(xì)胞沒有明顯的關(guān)系。將不同自交系的GBSSI過表達(dá)材料與突變型AGPase過表達(dá)材料進(jìn)行雜交,培育出總淀粉含量和直鏈淀粉高的轉(zhuǎn)基因玉米材料。ae突變體玉米與不同轉(zhuǎn)基因玉米的雜交,雖未得到聚合ae基因的純合材料,聚合種子的直鏈淀粉含量低于親本,但這為高直鏈淀粉玉米新種質(zhì)的創(chuàng)制提供了思路。轉(zhuǎn)野生型AGPase基因和轉(zhuǎn)突變型AGPase基因株系的雜交,聚合種子與親本相比具有增加的淀粉含量和百粒重。以上研究,為轉(zhuǎn)基因高淀粉、高直鏈淀粉玉米的研究提供了策略和方法,選育的高淀粉、高直鏈淀粉材料具有一定的應(yīng)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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S513

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