珙桐DiMYB1基因的克
發(fā)布時間：2018-07-24 12:08
【摘要】：珙桐(Davidia involucrata Baill.)為珙桐科珙桐屬落葉喬木,第三紀(jì)古熱帶植物孑遺種,我國特有的珍稀植物,國家I級重點保護(hù)瀕危物種,具有極高的觀賞價值和學(xué)術(shù)研究價值。本研究從珙桐果實及種子轉(zhuǎn)錄組數(shù)據(jù)庫中篩選到一個編碼調(diào)控原花青素生物合成的MYB轉(zhuǎn)錄因子的基因DiMYB1,對其進(jìn)行了克隆及功能分析。主要研究結(jié)果如下:1.珙桐DiMYB1基因的克隆及生物信息學(xué)分析。根據(jù)珙桐果實及種子轉(zhuǎn)錄組數(shù)據(jù)庫中該基因的ORF序列設(shè)計特異引物,以珙桐敗育種子的cDNA為模板進(jìn)行RCR擴(kuò)增得到全長序列,并在GenBank上登錄,登錄號為KR996175。利用在線軟件對該基因序列進(jìn)行了生物信息學(xué)分析。2.DiYB1基因的表達(dá)模式。①DiMYB1基因在珙桐芽、葉片、苞片、果肉、種子中的表達(dá)量qPCR分析。結(jié)果表明,DiMYB1基因在紫色幼葉中的表達(dá)量最高,其次是雄蕊,在芽、莖、成熟葉片、苞片、果肉和種子中微量表達(dá)。②DiMYB1基因在正常和敗育種子中的表達(dá)模式。qPCR分析結(jié)果表明,D/MYB1基因在敗育種子中的表達(dá)量顯著高于正常種子,且在中期敗育種子中的表達(dá)量達(dá)到最高。③DiMYB/基因在不同顏色葉片中的表達(dá)。DiMYB1基因在紫色葉片中的相對表達(dá)量最高,其次是中間色(介于紫色和淡黃綠色之間)葉片,在綠色葉片中表達(dá)量較低。3.珙桐DiMYB1基因的原核表達(dá)。構(gòu)建原核表達(dá)載體pET28a(+)-DiMYB1,高效表達(dá)蛋白質(zhì),采用His標(biāo)簽純化蛋白,DiMYB1蛋白以包涵體的形式存在。4.珙桐DiMYB1基因功能在擬南芥中的初步驗證。構(gòu)建植物超量表達(dá)載體pBI-121-DiMYB1,重組轉(zhuǎn)化農(nóng)桿菌菌株GV3101,采用花序浸染法轉(zhuǎn)化擬南芥花序,收獲種子后,通過抗性培養(yǎng)篩選陽性植株,自交繁殖2代后得到T2代轉(zhuǎn)基因植株。與野生型擬南芥相比,DiMYB1超量表達(dá)植株的主要表型差異表現(xiàn)在:大部分幼苗葉片顏色變紫;多數(shù)幼苗葉片的形態(tài)和數(shù)目發(fā)生了明顯變化,有的葉片出現(xiàn)了白化現(xiàn)象;少數(shù)成熟豆莢顏色變紫;有的豆莢不飽滿,種子有皺縮現(xiàn)象;植株矮小,莖稈細(xì)長,提前進(jìn)入生殖生長。后續(xù)將篩選純合子轉(zhuǎn)基因植株,驗證珙桐DiMB1基因的功能。
[Abstract]:Davidia involucrata (Davidia involucrata Baill.) It is a deciduous tree of the genus Davidia involucrata, a relict species of Tertiary paleotropical plants, a rare and rare plant endemic to China, and a national class I protected endangered species, which has very high ornamental value and academic research value. In this study, a gene DiMYB1 encoding MYB transcription factor regulating procyanidins biosynthesis was screened from the database of fruit and seed transcriptome of Davidia involucrata. The gene was cloned and its function was analyzed. The main results are as follows: 1. Cloning and bioinformatics analysis of DiMYB1 gene from Davidia involucrata. According to the ORF sequence of Davidia involucrata fruit and seed transcriptome database, a specific primer was designed. The full-length RCR sequence was amplified by using the cDNA of Davidia involucrata breeders as template, and the full-length sequence was registered on GenBank. The accession number was KR996175. The expression pattern of DiYB1 gene in bud, leaf, bract, pulp and seed of Davidia involucrata was analyzed with online software. 2. The expression level of DiYB1 gene in bud, leaf, bract, pulp and seed was analyzed by qPCR. The results showed that DiMYB1 gene expression was the highest in young purple leaves, followed by stamens, buds, stems, mature leaves and bracts. The expression pattern of DiMYB1 gene in both normal and abortive breeders was analyzed by qPCR. The results showed that the expression of D _ (r) MYB1 gene in abortive breeders was significantly higher than that in normal seeds. The highest expression level of .3DiMYB/ gene was found in different color leaves, and the relative expression of DiMYB1 gene was the highest in purple leaves, followed by intermediate color leaves (between purple and yellowish green). The expression level in green leaves was lower. 3. Prokaryotic expression of DiMYB1 gene in Davidia involucrata. The prokaryotic expression vector pET28a () -DiMYB1 was constructed and the protein was highly expressed. The protein was purified by His tag and existed in the form of inclusion body. The function of Davidia involucrata DiMYB1 gene in Arabidopsis thaliana was preliminarily verified. The plant overexpression vector pBI-121-DiMYB1 was constructed and transformed into Agrobacterium tumefaciens strain GV3101. The inflorescence was transformed into Arabidopsis thaliana by inflorescence soaking. After harvesting the seeds, the positive plants were screened by resistance culture, and the transgenic plants of T2 generation were obtained after 2 generations of self-breeding. Compared with wild type Arabidopsis thaliana, the main phenotypic differences of DiMYB1 overexpression plants were as follows: the color of most seedling leaves became purple, the morphology and number of most seedling leaves changed obviously, and some leaves appeared albinism. A few mature pods became purple in color; some of them were not full and the seeds were crumpled; the plants were short and the stems were slender and entered into reproductive growth ahead of time. The homozygous transgenic plants will be screened to verify the function of DiMB1 gene in Davidia involucrata.
【學(xué)位授予單位】：中南林業(yè)科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S792.99;Q943.2
，
本文編號：2141333
[Abstract]:Davidia involucrata (Davidia involucrata Baill.) It is a deciduous tree of the genus Davidia involucrata, a relict species of Tertiary paleotropical plants, a rare and rare plant endemic to China, and a national class I protected endangered species, which has very high ornamental value and academic research value. In this study, a gene DiMYB1 encoding MYB transcription factor regulating procyanidins biosynthesis was screened from the database of fruit and seed transcriptome of Davidia involucrata. The gene was cloned and its function was analyzed. The main results are as follows: 1. Cloning and bioinformatics analysis of DiMYB1 gene from Davidia involucrata. According to the ORF sequence of Davidia involucrata fruit and seed transcriptome database, a specific primer was designed. The full-length RCR sequence was amplified by using the cDNA of Davidia involucrata breeders as template, and the full-length sequence was registered on GenBank. The accession number was KR996175. The expression pattern of DiYB1 gene in bud, leaf, bract, pulp and seed of Davidia involucrata was analyzed with online software. 2. The expression level of DiYB1 gene in bud, leaf, bract, pulp and seed was analyzed by qPCR. The results showed that DiMYB1 gene expression was the highest in young purple leaves, followed by stamens, buds, stems, mature leaves and bracts. The expression pattern of DiMYB1 gene in both normal and abortive breeders was analyzed by qPCR. The results showed that the expression of D _ (r) MYB1 gene in abortive breeders was significantly higher than that in normal seeds. The highest expression level of .3DiMYB/ gene was found in different color leaves, and the relative expression of DiMYB1 gene was the highest in purple leaves, followed by intermediate color leaves (between purple and yellowish green). The expression level in green leaves was lower. 3. Prokaryotic expression of DiMYB1 gene in Davidia involucrata. The prokaryotic expression vector pET28a () -DiMYB1 was constructed and the protein was highly expressed. The protein was purified by His tag and existed in the form of inclusion body. The function of Davidia involucrata DiMYB1 gene in Arabidopsis thaliana was preliminarily verified. The plant overexpression vector pBI-121-DiMYB1 was constructed and transformed into Agrobacterium tumefaciens strain GV3101. The inflorescence was transformed into Arabidopsis thaliana by inflorescence soaking. After harvesting the seeds, the positive plants were screened by resistance culture, and the transgenic plants of T2 generation were obtained after 2 generations of self-breeding. Compared with wild type Arabidopsis thaliana, the main phenotypic differences of DiMYB1 overexpression plants were as follows: the color of most seedling leaves became purple, the morphology and number of most seedling leaves changed obviously, and some leaves appeared albinism. A few mature pods became purple in color; some of them were not full and the seeds were crumpled; the plants were short and the stems were slender and entered into reproductive growth ahead of time. The homozygous transgenic plants will be screened to verify the function of DiMB1 gene in Davidia involucrata.
【學(xué)位授予單位】：中南林業(yè)科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S792.99;Q943.2
，
本文編號：2141333
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