本文選題：天然橡膠 + 乙烯利處理��； 參考：《海南大學(xué)》2017年碩士論文

【摘要】：天然橡膠(順式-1,4-聚異戊二稀)是四大工業(yè)原料之一和極其重要的戰(zhàn)略資源。橡膠樹(shù)目前是天然橡膠的最主要來(lái)源。乙烯利(一種乙烯釋放劑)處理橡膠樹(shù)樹(shù)皮是增加乳膠產(chǎn)量的一種常規(guī)措施,但乙烯刺激膠乳增產(chǎn)的分子機(jī)制仍然是一個(gè)謎。破解這個(gè)謎對(duì)提高橡膠產(chǎn)量具有重要意義。橡膠樹(shù)天然橡膠生物合成是通過(guò)甲羥戊酸(MVA)途徑進(jìn)行的。3-羥基-3-甲基戊二酸單酰輔酶A還原酶(HMGR)被認(rèn)為是MVA途徑中的第一個(gè)限速酶。HMGR為一個(gè)小的基因家族,其中HMGR1主要在橡膠樹(shù)乳汁管中表達(dá),參與天然橡膠生物合成。通過(guò)對(duì)HMGR1基因啟動(dòng)子的生物信息學(xué)分析和缺失片段轉(zhuǎn)化表達(dá)分析,可鑒定控制HMGR1基因啟動(dòng)子的關(guān)鍵調(diào)控元件,了解對(duì)HMGR1基因表達(dá)的調(diào)控因素。對(duì)乙烯利處理橡膠樹(shù)樹(shù)皮轉(zhuǎn)錄組分析與IPP限速酶基因HMGR1啟動(dòng)子分析將會(huì)增進(jìn)對(duì)天然橡膠生物合成調(diào)控機(jī)理的了解,有助于利用激素或相關(guān)活性物質(zhì)刺激膠乳增產(chǎn)。1分別對(duì)用乙烯利處理8小時(shí)和24小時(shí)的橡膠樹(shù)皮進(jìn)行RNA從頭測(cè)序和組裝。E8(乙烯利處理8小時(shí))、E24(乙烯利處理24小時(shí))和C(對(duì)照組)分別得到51965770、52303714 和 53177976 個(gè)高品質(zhì)的 clear reads,然后分別組裝成 81335、80048 和 80800個(gè)unigenes,共獲得84425個(gè)平均長(zhǎng)度為1101bp的unigenes。與C相比,E8和E24分別檢測(cè)到10216個(gè)和9374個(gè)差異表達(dá)基因(DEGs)。發(fā)現(xiàn)調(diào)節(jié)糖酵解途徑中的關(guān)鍵酶基因表達(dá)上調(diào),而異戊烯基焦磷酸(IPP)的生物合成途徑差異基因并未顯著富集。此外,鑒定到碳固定(卡爾文循環(huán))中的一些重要的調(diào)控基因是上調(diào)的。由此可見(jiàn),糖酵解途徑加速運(yùn)行以提供充足的IPP和天然橡膠生物合成前體,同時(shí)抑制乙酰輔酶A流向乙醇合成,而不是影響橡膠生物合成途徑本身,是乙烯刺激橡膠樹(shù)膠乳增產(chǎn)的主要原因。樹(shù)皮卡爾文循環(huán)通量提高會(huì)源源不斷地提供碳水化合物以供膠乳再生,從而增加乙烯刺激效果的持久性。2通過(guò)PCR方法從橡膠樹(shù)熱研7-33-97的葉片DNA中分離到了HMGR 基因啟動(dòng)子。HMGR1基因啟動(dòng)子序列長(zhǎng)度為1460bp。使用PlantCARE在線(xiàn)預(yù)測(cè)該啟動(dòng)子除了含有TATA-box、CAAT-box、增強(qiáng)子等基本順式作用元件外,還含有2個(gè)茉莉酸相關(guān)元件、3個(gè)乙烯相關(guān)元件、1個(gè)脫落酸相關(guān)元件、一些水楊酸相關(guān)元件等激素響應(yīng)元件,熱脅迫相關(guān)元件、厭氧誘導(dǎo)相關(guān)元件等與抗性脅迫相關(guān)的順式作用元件及光反應(yīng)相關(guān)元件、胚乳誘導(dǎo)相關(guān)元件等元件。這顯示橡膠樹(shù)HMGR1基因的啟動(dòng)子很可能是一個(gè)組織特異型和誘導(dǎo)型啟動(dòng)子,并可能在橡膠樹(shù)的生長(zhǎng)發(fā)育和逆境脅迫應(yīng)答過(guò)程中發(fā)揮調(diào)控作用。3構(gòu)建橡膠樹(shù)HMGR1基因啟動(dòng)子序列連接報(bào)告基因GUS(β-葡萄糖苷酸酶基因)的植物表達(dá)載體P1301-H1-1460,并將其轉(zhuǎn)入模式植物擬南芥中。PCR檢測(cè)表明以GUS為報(bào)告基因的植物表達(dá)載體P1301-H1-1460成功轉(zhuǎn)入擬南芥。RT-PCR檢測(cè)表明以GUS為報(bào)告基因的植物表達(dá)載體P1301-H1-1460可在擬南芥中轉(zhuǎn)錄。4對(duì)P1301-H1-1460轉(zhuǎn)化的擬南芥兩周大幼苗噴施茉莉酸甲酯、乙稀利等外源激素,分別取處理0h、3 h、6 h、9 h、12 h、24 h等不同時(shí)間的幼苗進(jìn)行GUS的實(shí)時(shí)熒光定量PCR以檢測(cè)其表達(dá)水平,結(jié)果發(fā)現(xiàn)不同時(shí)間的MeJA處理下GUS的表達(dá)量均明顯比未處理的轉(zhuǎn)基因苗低;不同時(shí)間的乙稀利處理的GUS表達(dá)量在一定時(shí)間范圍內(nèi)先增加后降低。表明,HMGR1基因啟動(dòng)子屬于誘導(dǎo)型啟動(dòng)子,外源激素MeJA對(duì)橡膠樹(shù)HMGR1基因啟動(dòng)子轉(zhuǎn)錄水平起下調(diào)作用,乙稀利對(duì)橡膠樹(shù)HMGR1基因啟動(dòng)子轉(zhuǎn)錄水平在一定時(shí)間內(nèi)起上調(diào)作用。5此外,還采用5'端序列缺失的方法,構(gòu)建P1301-H1-1290,P1301-H1-894,P1301-H1-593,P1301-H1-444 和 P1301-H1-188 等橡膠樹(shù)HMGR 基因啟動(dòng)子 5' 端缺失表達(dá)載體并分別轉(zhuǎn)入擬南芥中,通過(guò)對(duì)比各轉(zhuǎn)入5'端缺失表達(dá)載體的擬南芥的GUS表達(dá)情況來(lái)分析橡膠樹(shù)HMGR1基因啟動(dòng)子不同片段對(duì)啟動(dòng)子活性的影響。
[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.
【學(xué)位授予單位】：海南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q943.2;S794.1

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