小麦春化相关基因的克隆及其功能研究

发布时间：2018-10-24 15:20

【摘要】：春化阶段是小麦生长发育进程中一个及其关键的时期。本研究从不同春化发育特性小麦辽春10和京841春化响应转录组Illumina高通量测序结果中,筛选出2个强烈上调的ESTs CL13519、Unigene 1312和2个下调ESTs Unigene 4460、Unigene 20。以春性小麦辽春10和冬性小麦京841为材料,系统分析了4个候选ESTs在转录水平的序列差异及春化处理过程中的表达特性,利用病毒诱导的基因沉默技术进行基因功能验证;构建CL13519过表达载体,利用农杆菌介导法将重组载体导入拟南芥中,分析转基因材料的生长发育特性。主要研究结果如下:1、以春性品种辽春10号和冬性品种京841为材料,克隆并分析4个ESTs的序列差异。结果显示:CL13519长1010 bp,ORF长681 bp,编码226个氨基酸,与Ta HOX1(Gen Bank:JQ915062.1)相似度87.49%;Unigene1312长度为479 bp,ORF全长282 bp,5′UTR 169 bp,3′UTR 26 bp,编码93个氨基酸,与硬质四倍体小麦脱水素(Gen Bank:X78432.1)一致性高达85%;Unigene4460长361bp,经TAIR在线分析,可能与H3蛋白甲基化相关;Unigene20长632 bp,与中国春3B染色体上一段序列(Gen Bank:HG670306.1)相似度高达83.91%,与山羊草hypothetical protein F775_32582序列(Gen Bank:KD510223.1)相似度达77.15%。2、利用实时荧光定量PCR分析了4个ESTs在不同春化特性小麦中的表达特性,结果表明:CL13519、Unigene1312在辽春10号中表达量呈抛物线型表达,第5周时表达量达到最大值;Unigene1312在京841中的表达量呈抛物线型表达,第5周时表达量达到最大值;CL13519在京841中表达模式中有两个峰值,最大值出现在春化处理第五周;Unigene4460在辽春10号中表达量呈波动式下调表达;Unigene4460在京841中第1周急剧升高后急剧下降之后一直下调表达;Unigene20在辽春10号和京841中均呈波浪型表达,在第5周时表达量最低。3、以辽春10号为试验材料,利用病毒诱导的基因沉默技术(VIGS),成功构建了小麦VIGS基因功能验证的技术体系,并对所挑选的4个ESTs进行验证分析。结果显示:BSMV的侵染对小麦植株幼穗的分化进程产生了一定的影响。本试验中空白对照组、阴性对照组、阳性对照组之间小麦幼穗分化几乎无差异,BSMV:CL13519、BSMV:Unigene1312、BSMV:Unigene20幼穗分化发育晚于对照,而BSMV:Unigene4460侵染却加速了幼穗分化。4、以pCAMBIA3301为表达载体成功构建CL13519过表达载体,并通过农杆菌介导法对拟南芥进行转化,通过标记基因进行除草剂抗性筛选以及PCR鉴定,得到阳性转基因T1代植株,经分析发现T1代转基因植株营养生长、生殖生长早于野生型植株。
[Abstract]:The vernalization stage is an important stage in wheat growth and development. In this study, two highly up-regulated ESTs CL13519,Unigene 1312 and two down-regulated ESTs Unigene 4460 Unigene 20 were screened from the high-throughput Illumina sequencing results of the vernalization response transcriptional sets of wheat Liaochun 10 and Jing841 with different vernalization developmental characteristics. Using the spring wheat Liaochun 10 and winter wheat Jing841 as materials, the sequence differences of four candidate ESTs at transcription level and their expression characteristics during vernalization were systematically analyzed, and the gene function was verified by virus-induced gene silencing. CL13519 overexpression vector was constructed and the recombinant vector was introduced into Arabidopsis thaliana by Agrobacterium tumefaciens to analyze the growth and development characteristics of transgenic materials. The main results are as follows: 1. Using the spring variety Liaochun 10 and winter variety Jing841 as materials, the sequence differences of four ESTs were cloned and analyzed. The results showed that CL13519 length 1010 bp,ORF length 681 bp, encode 226 amino acids, and the similarity with Ta HOX1 (Gen Bank:JQ915062.1) is 87.49%. The length of bp,ORF is 282 bp,5'UTR 169 bp,3'UTR 26 bp, which encodes 93 amino acids. The consistency with hard tetraploid wheat dehydrin (Gen Bank:X78432.1) is as high as 851 BP by TAIR on-line analysis. The similarity between Unigene20 length 632 bp, and Chinese Spring 3B chromosome (Gen Bank:HG670306.1) is as high as 83.91l, and the similarity with hypothetical protein F775Serial 32582 (Gen Bank:KD510223.1) is 77.150.The four ESTs were analyzed by real-time fluorescence quantitative PCR. Vernalization characteristics in wheat, The results showed that the expression of CL13519,Unigene1312 was parabolic in Liaochun 10, and reached the maximum at the 5th week, and the expression of Unigene1312 in Jing841 was parabola. At the 5th week, the expression of CL13519 reached its maximum value, and there were two peaks in the expression pattern of CL13519 in Jing841. The maximum value appeared in the fifth week of vernalization, the expression of Unigene4460 in Liaochun 10 was down-regulated, the expression of Unigene4460 in Jing841 increased sharply in the first week and then decreased sharply, and the expression of Unigene20 was wave-like in Liaochun 10 and Jing841. At the fifth week, the lowest expression was found. 3. Using Liaochun 10 as the experimental material, the functional verification system of wheat VIGS gene was successfully constructed by virus induced gene silencing technique (VIGS), and the four selected ESTs were verified and analyzed. The results showed that BSMV infection had a certain effect on the process of spike differentiation of wheat plants. In this experiment, there was almost no difference between the blank control group, the negative control group and the positive control group. The spike differentiation of BSMV:CL13519,BSMV:Unigene1312,BSMV:Unigene20 was later than that of the control, but the BSMV:Unigene4460 infection accelerated the spike differentiation. 4. The CL13519 overexpression vector was successfully constructed with pCAMBIA3301 as expression vector. Through Agrobacterium tumefaciens mediated transformation of Arabidopsis thaliana, herbicide resistance screening and PCR identification of marker genes, positive transgenic T1 plants were obtained. Reproductive growth was earlier than wild-type plants.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S512.1

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