Construction of wheat starch branching enzyme sbeⅡb gene expression vector and its expression identification | 小麦籽粒胚乳中淀粉分支酶基因的RNAi表达载体的构建及表达
2011
Wang Zibu, Shihezi University,Shihezi(China) | Qi Juncang, Shihezi University,Shihezi(China) | Li Weihua, Shihezi University,Shihezi(China)
Chinese. 摘 要：构建sbe2b的RNAi表达载体,并对其转化烟草,检测其对直链淀粉合成的影响。利用网络数据库http：//jura.wi.mit.edu/bioc/siRNAext/,确定小麦胚乳淀粉分支酶sbe2b的部分基因序列作为RNAi的目标序列,通过RT-PCR方法从小麦籽粒胚乳中成功克隆出了小麦胚乳中淀粉分支酶sbe2b基因的部分序列（300 bp）（GenBank No.AY740401）,以植物表达载体PZP35S为基础,构建了由组成型启动子35S调控的sbe2b基因的RNAi植物表达载体。实时荧光定量PCR（qRT-PCR）检测其在转基因烟草中的相对表达量,并测定转基因植株中直链淀粉的含量和支链淀粉含量的变化。结果表明：经qRT-PCR证实sbe2b基因的表达量较对照下降,而且支链淀粉含量变化显著,直链淀粉较对照有上升但变化没有达到显著水平。
Show more [+] Less [-]English. To obstain RNAi expressive vector of sbe2b gene,this integrated in tobacco genome and studies its effect on amylase content.As the targeted sequences of RNAi,parts of sbe2b gene starch brangching enzyme in maize was ascertained by searching internet database http：//jura.wi.mit.edu/bioc/siRNAext/.Segments of sbe2b gene（300 bp）（GenBank No.AY740401） from wheat endosperm were cloned separately by RT-PCR.RNAi vector was constructed with PZP35S and pUCm-T,and Expression vector RNAi containing 35S promoter to regulate gene expression.qRT-PCR analysis results indicated that the SBE2b gene expression value is lower than CK（check plants） in the leaves of all transformed plants.Comparing with the CK about starch content,the amylopectin content of the transformed plants reached significantly changes,however,the amylose showed no significant difference.
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