本文選題：水稻 + PULL基因�。� 參考：《揚(yáng)州大學(xué)》2016年碩士論文

【摘要】：淀粉是稻米胚乳的主要成分,在一定程度上決定了稻米的品質(zhì),淀粉合成是通過多種酶協(xié)同催化完成,其中淀粉去分支酶(Starch debranching enzyme,DBE)對淀粉最終結(jié)構(gòu)的形成起著非常重要的作用,因此,對DBE的深入研究有助于解釋淀粉生物合成機(jī)理。淀粉去分支酶包括普魯蘭酶(PULL)和異淀粉酶(ISA),異淀粉酶又包括ISA1、ISA2、ISA3三種同工型。本研究利用根癌農(nóng)桿菌介導(dǎo)的轉(zhuǎn)基因技術(shù)將PULL基因RNA干擾結(jié)構(gòu)和ISA3基因RNA干擾結(jié)構(gòu)導(dǎo)入受體品種日本晴中,且從2類干擾材料的雜交后代中通過PCR篩選到不同轉(zhuǎn)化子來源的雙基因干擾純系。本實(shí)驗(yàn)中所用到的材料有的PULL和ISA3雙干擾株系(PULL/ISA3-RNAi)四個,PULL干擾株系(PULL-RNAi)4個,ISA3干擾株系(ISA3-RNAi)3個和一個未轉(zhuǎn)化對照。對獲得的雙干擾純系和其親本,以及未轉(zhuǎn)化對照進(jìn)行表達(dá)量分析,并測定稻米淀粉的理化特性和結(jié)構(gòu)特性,以研究PULL、ISA3基因?qū)Φ久灼焚|(zhì)的影響。主要研究結(jié)果如下：(1)對雙干擾材料及相關(guān)親本水稻胚乳總RNA的Real-time PCR分析結(jié)果表明,單基因干擾材料中,PULL或ISA3基因的表達(dá)量不同程度的降低,且達(dá)到極顯著水平。雙基因干擾材料中PULL和ISA3的表達(dá)量與其對應(yīng)的單基因干擾親本材料相比無顯著性差異。表明RNAi結(jié)構(gòu)均已經(jīng)發(fā)揮了作用,成功的抑制了相關(guān)基因的表達(dá)。(2)理化分析表明尸ULL/ISA3-RNAi,PULL-RNAi和ISA3-RNAi材料的染色范圍和染色程度無明顯的差別。轉(zhuǎn)基因植株胚乳總淀粉含量、直鏈淀粉含量和可溶性總糖含量和未轉(zhuǎn)化對照無明顯差異,表明PULL或ISA3基因的低表達(dá)沒有改變胚乳中淀粉分布和植物糖原分布。(3)對轉(zhuǎn)基因材料的稻米淀粉RVA測定和DSC測定結(jié)果顯示,轉(zhuǎn)基因材料RVA和DSC的特征值與對照相比沒有達(dá)到顯著性差異。(4)對轉(zhuǎn)基因材料的稻米淀粉進(jìn)行X-射線衍射和紅外光譜全反射分析,結(jié)果表明PULL/ISA3-RNAi,PULL-RNAi和ISA3-RNAi材料淀粉粒的晶型和結(jié)晶度無明顯變化。(5)轉(zhuǎn)基因株系稻米淀粉精細(xì)結(jié)構(gòu)分析表明,PULL-RNAi和ISA3-RNAi株系中10≤DP≤20的淀粉鏈較未轉(zhuǎn)化對照略微減少,大部分PULL/ISA3-RNAi材料胚乳中支鏈淀粉中10≤DP≤35的淀粉鏈均比單基因干擾材料含量高,說明PULL,ISA3都影響了支鏈淀粉的精細(xì)結(jié)構(gòu)。(6)對轉(zhuǎn)基因水稻農(nóng)藝性狀調(diào)查發(fā)現(xiàn),PULL/ISA3-RNAi材料和單基因干擾材料的有效分蘗數(shù)、主穗長、結(jié)實(shí)率整體上和未轉(zhuǎn)化對照無明顯差異,植株株高和結(jié)實(shí)率也沒有明顯的規(guī)律性變化。
[Abstract]:Starch is the main component of rice endosperm, which determines the quality of rice to a certain extent. Starch synthesis is catalyzed by a variety of enzymes, among which starch debranching enzyme DBE plays a very important role in the formation of starch final structure. Therefore, the in-depth study of DBE is helpful to explain the mechanism of starch biosynthesis. Starch debranching enzymes include PULL) and isoamylase ISAA, and isoamylase include ISA1, ISA2 and ISA3 isoforms. In this study, the RNA interference structure of call gene and ISA3 gene RNA interference structure were introduced into the recipient variety Nippon by Agrobacterium tumefaciens mediated transgenic technology. Double gene interference pure lines from different transformants were screened by PCR from hybrid progenies of two kinds of interference materials. The materials used in this experiment were pul and ISA3 double interference lines (PULL / ISA3-RNAi), PULL / ISA3-RNAi4 (PULL-RNAi3), ISA3-RNAi3 (3) and one untransformed control. In order to study the effect of PULLN ISA3 gene on rice quality, the expression of double interference pure lines, their parents and untransformed control were analyzed, and the physicochemical and structural characteristics of rice starch were determined. The main results were as follows: (1) Real-time PCR analysis of total endosperm RNA of two interfering materials and their related parents showed that the expression of pail or ISA3 gene in single gene interference materials decreased in varying degrees and reached an extremely significant level. There was no significant difference in the expression of pul and ISA3 between the two gene interference materials and their monogenic interference parents. The results of physicochemical analysis showed that there was no significant difference in staining range and staining degree between PULL / ISA3-RNAi and ISA3-RNAi in PULL-RNAi and ISA3-RNAi. There was no significant difference in total starch content, amylose content and soluble sugar content between transgenic plants and untransformed control. The results showed that the low expression of pul or ISA3 gene did not change the starch distribution in endosperm and the distribution of plant glycogen. The characteristic values of RVA and DSC were not significantly different from those of the control. The results showed that the grain shape and crystallinity of starch granules of PULL / ISA3-RNAi and ISA3-RNAi were not significantly changed. The results of starch fine structure analysis showed that the starch chains of PULL-RNAi and ISA3-RNAi lines with 10 鈮，

本文編號：2000414