橡胶树皮乙烯利处理转录组分析与HMGR1基因启动子克隆分析

发布时间：2018-06-12 19:26

本文选题：天然橡胶 + 乙烯利处理　；参考：《海南大学》2017年硕士论文

【摘要】：天然橡胶(顺式-1,4-聚异戊二稀)是四大工业原料之一和极其重要的战略资源。橡胶树目前是天然橡胶的最主要来源。乙烯利(一种乙烯释放剂)处理橡胶树树皮是增加乳胶产量的一种常规措施,但乙烯刺激胶乳增产的分子机制仍然是一个谜。破解这个谜对提高橡胶产量具有重要意义。橡胶树天然橡胶生物合成是通过甲羟戊酸(MVA)途径进行的。3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGR)被认为是MVA途径中的第一个限速酶。HMGR为一个小的基因家族,其中HMGR1主要在橡胶树乳汁管中表达,参与天然橡胶生物合成。通过对HMGR1基因启动子的生物信息学分析和缺失片段转化表达分析,可鉴定控制HMGR1基因启动子的关键调控元件,了解对HMGR1基因表达的调控因素。对乙烯利处理橡胶树树皮转录组分析与IPP限速酶基因HMGR1启动子分析将会增进对天然橡胶生物合成调控机理的了解,有助于利用激素或相关活性物质刺激胶乳增产。1分别对用乙烯利处理8小时和24小时的橡胶树皮进行RNA从头测序和组装。E8(乙烯利处理8小时)、E24(乙烯利处理24小时)和C(对照组)分别得到51965770、52303714 和 53177976 个高品质的 clear reads,然后分别组装成 81335、80048 和 80800个unigenes,共获得84425个平均长度为1101bp的unigenes。与C相比,E8和E24分别检测到10216个和9374个差异表达基因(DEGs)。发现调节糖酵解途径中的关键酶基因表达上调,而异戊烯基焦磷酸(IPP)的生物合成途径差异基因并未显著富集。此外,鉴定到碳固定(卡尔文循环)中的一些重要的调控基因是上调的。由此可见,糖酵解途径加速运行以提供充足的IPP和天然橡胶生物合成前体,同时抑制乙酰辅酶A流向乙醇合成,而不是影响橡胶生物合成途径本身,是乙烯刺激橡胶树胶乳增产的主要原因。树皮卡尔文循环通量提高会源源不断地提供碳水化合物以供胶乳再生,从而增加乙烯刺激效果的持久性。2通过PCR方法从橡胶树热研7-33-97的叶片DNA中分离到了HMGR 基因启动子。HMGR1基因启动子序列长度为1460bp。使用PlantCARE在线预测该启动子除了含有TATA-box、CAAT-box、增强子等基本顺式作用元件外,还含有2个茉莉酸相关元件、3个乙烯相关元件、1个脱落酸相关元件、一些水杨酸相关元件等激素响应元件,热胁迫相关元件、厌氧诱导相关元件等与抗性胁迫相关的顺式作用元件及光反应相关元件、胚乳诱导相关元件等元件。这显示橡胶树HMGR1基因的启动子很可能是一个组织特异型和诱导型启动子,并可能在橡胶树的生长发育和逆境胁迫应答过程中发挥调控作用。3构建橡胶树HMGR1基因启动子序列连接报告基因GUS(β-葡萄糖苷酸酶基因)的植物表达载体P1301-H1-1460,并将其转入模式植物拟南芥中。PCR检测表明以GUS为报告基因的植物表达载体P1301-H1-1460成功转入拟南芥。RT-PCR检测表明以GUS为报告基因的植物表达载体P1301-H1-1460可在拟南芥中转录。4对P1301-H1-1460转化的拟南芥两周大幼苗喷施茉莉酸甲酯、乙稀利等外源激素,分别取处理0h、3 h、6 h、9 h、12 h、24 h等不同时间的幼苗进行GUS的实时荧光定量PCR以检测其表达水平,结果发现不同时间的MeJA处理下GUS的表达量均明显比未处理的转基因苗低;不同时间的乙稀利处理的GUS表达量在一定时间范围内先增加后降低。表明,HMGR1基因启动子属于诱导型启动子,外源激素MeJA对橡胶树HMGR1基因启动子转录水平起下调作用,乙稀利对橡胶树HMGR1基因启动子转录水平在一定时间内起上调作用。5此外,还采用5'端序列缺失的方法,构建P1301-H1-1290,P1301-H1-894,P1301-H1-593,P1301-H1-444 和 P1301-H1-188 等橡胶树HMGR 基因启动子 5' 端缺失表达载体并分别转入拟南芥中,通过对比各转入5'端缺失表达载体的拟南芥的GUS表达情况来分析橡胶树HMGR1基因启动子不同片段对启动子活性的影响。
[Abstract]:Natural rubber (CIS -1,4- polyamyl two) is one of the four major industrial raw materials and extremely important strategic resources. The rubber tree is currently the main source of natural rubber. Ethephon (a ethylene releasing agent) is a conventional measure to increase the yield of latex, but the molecular mechanism of ethylene stimulation is still one of the molecular mechanisms. The riddle is important to improve the rubber production. The rubber tree natural rubber biosynthesis is the.3- hydroxyl -3- methylglutaric acid monoyl coenzyme A reductase (HMGR) based on the MVA pathway (HMGR), which is considered the first speed limiting enzyme.HMGR in the MVA pathway as a small gene family, in which HMGR1 is mainly in the rubber tree. The milk tube is expressed in the natural rubber biosynthesis. Through the bioinformatics analysis of the HMGR1 gene promoter and the analysis of the missing fragment transformation and expression analysis, the key regulatory elements to control the HMGR1 gene promoter can be identified and the regulation factors for the expression of HMGR1 gene are understood. The analysis of the bark transcriptional group of the ethephon tree bark and the IPP speed limiting enzyme The gene HMGR1 promoter analysis will enhance the understanding of the regulation mechanism of natural rubber biosynthesis, and help to stimulate the production of.1 by hormone or related active substances to increase the yield of rubber, and to carry out RNA ab initio sequencing and assembly of.E8 (8 hours of vinyl treatment), E24 (ethephon treatment 24 hours) and C, respectively, for the treatment of the rubber bark of the rubber with Ethephon for 8 hours and 24 hours respectively. (control group) 5196577052303714 and 53177976 high quality clear reads were obtained, then 8133580048 and 80800 unigenes were assembled respectively, and 84425 unigenes. with an average length of 1101bp were compared with C, E8 and E24 detected 10216 and 9374 differentially expressed genes (DEGs). The gene expression of the key enzyme is up, and the biosynthesis pathway of Isoamyl pyrophosphate (IPP) is not significantly enriched. Furthermore, some important regulatory genes in the carbon fixation (Calvin cycle) are up regulated. Thus, the glycolytic pathway is accelerated to provide sufficient IPP and natural rubber biosynthesis precursors, as well. The inhibition of acetyl coenzyme A to ethanol synthesis, rather than the effect of rubber biosynthesis itself, is the main reason for increasing production of rubber tree latex by ethylene. The increase of Calvin's bark flux will continuously provide carbohydrates for latex regeneration, thus increasing the persistence of ethylene stimulation effect by the PCR method from the rubber tree. The HMGR gene promoter.HMGR1 promoter sequence length of the HMGR gene promoter was separated by the HMGR gene promoter.HMGR1 gene promoter sequence, using PlantCARE online to predict the promoter in addition to the basic cis element containing TATA-box, CAAT-box, and enhancers, and also contained 2 jasmonic acid related elements, 3 ethylene related elements, and 1 abscisic acid related elements. Some elements such as salicylic acid related elements, such as hormone response elements, heat stress related elements, anaerobic induction related elements, and other elements related to resistance stress, such as cis acting elements and light reaction related elements, endosperm induction components, etc., show that the promoter of the HMGR1 gene of the rubber tree is probably a tissue specific and inducible promoter. In the process of growth and stress response of the rubber tree,.3 can play a regulatory role in the construction of the plant expression vector P1301-H1-1460 of the HMGR1 gene promoter GUS (beta glucosidase gene) of the rubber tree, and transfer it into the model plant Arabidopsis thaliana to detect the expression of plant expression with GUS as the reporter gene. The carrier P1301-H1-1460 successfully transferred to Arabidopsis.RT-PCR test that the plant expression vector P1301-H1-1460 with GUS as the reporter gene could transcribe.4 into the Arabidopsis thaliana with.4 and spraying jasmonate, ethylene and other exogenous hormones in Arabidopsis thaliana seedlings transformed by P1301-H1-1460, and take the seedlings of 0h, 3 h, 6 h, 9 h, 12 h, 24 h, and so on. GUS real-time fluorescence quantitative PCR was used to detect its expression level. The results showed that the expression of GUS under MeJA treatment at different time was significantly lower than that of the untreated transgenic seedlings. The GUS expression of GUS at different time increased and then decreased in a certain time range. It showed that the HMGR1 gene promoter was an inducible promoter, and the exogenous gene was the inducible promoter. Hormone MeJA regulates the transcriptional level of the HMGR1 gene promoter in the rubber tree. The transcriptional level of the HMGR1 gene promoter of the rubber tree is up regulating.5 in a certain time. Furthermore, the 5'end sequence deletion method is used to construct P1301-H1-1290, P1301-H1-894, P1301-H1-593, P1301-H1-444, P1301-H1-188 and other rubber tree HMGR bases. The effect of different fragments of the HMGR1 gene promoter on the promoter activity of the rubber tree HMGR1 gene was analyzed by comparing the GUS expression of the Arabidopsis thaliana by comparing the expression of the Arabidopsis thaliana which was transferred to the deletion expression vector of the 5'terminal in the Arabidopsis, the deletion expression vector of the promoter was transferred to the Arabidopsis.
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S794.1

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