高粱SbbHLH1互作蛋白、DNA結(jié)合序列的識別及毛白楊轉(zhuǎn)基因的初步研究
發(fā)布時間：2018-11-25 15:38
【摘要】：高粱是重要的能源作物之一,其bmr突變體表現(xiàn)出低木質(zhì)素含量。閆麗(2011)等利用以高粱野生型BTx623作為tester、以13種不同的bmr突變體作為driver構(gòu)建的抑制差減雜交(SSH)文庫,通過芯片雜交識別出大量轉(zhuǎn)錄差異的基因。SbbHLH1和SbLIM1是其中兩個差異較明顯的轉(zhuǎn)錄因子基因。前期的研究發(fā)現(xiàn),SbbHLH1和SbLIM1影響擬南芥的木質(zhì)素生物合成。本文對SbbHLH1調(diào)控木質(zhì)素合成代謝機(jī)制及毛白楊的遺傳轉(zhuǎn)化進(jìn)行初步的研究,探討這兩個轉(zhuǎn)錄因子對其木質(zhì)素合成的影響。采用酵母雙雜技術(shù)可以研究與SbbHLH1轉(zhuǎn)錄因子的互作蛋白,結(jié)果發(fā)現(xiàn)SbbHLH1轉(zhuǎn)錄因子可以與高粱中的SB02G040140蛋白(也是一個bHLH類的蛋白)互作。表明了SbbHLH1轉(zhuǎn)錄因子會與其他的bHLH結(jié)構(gòu)域蛋白結(jié)合,再參與到木質(zhì)素的轉(zhuǎn)錄調(diào)控網(wǎng)絡(luò)。采用SELEX技術(shù)分離得到了與SbbHLH1轉(zhuǎn)錄因子結(jié)合的DNA基序E-box (CANNTG),為了驗(yàn)證SbbHLH1轉(zhuǎn)錄因子能夠結(jié)合擬南芥木質(zhì)素生物合成途徑相關(guān)基因啟動子序列中的E-box基序,克隆這些序列,通過DNA和蛋白的結(jié)合實(shí)驗(yàn)發(fā)現(xiàn)SbbHLH1轉(zhuǎn)錄因子可以與AtPAL基因的啟動子結(jié)合,這就進(jìn)一步證明了SbbHLH1轉(zhuǎn)錄因子在擬南芥中可以通過結(jié)合PAL基因調(diào)控木質(zhì)素的生物合成。用攜帶載體pSTART-SbLIM1的農(nóng)桿菌侵染毛白楊芽尖的外植體,通過組織培養(yǎng)、卡那霉素篩選、PCR鑒定獲得12株陽性植株。同時對三年生SbbHLH1轉(zhuǎn)基因毛白楊及SbLIM1轉(zhuǎn)基因毛白楊的木質(zhì)素含量進(jìn)行測定,結(jié)果發(fā)現(xiàn)轉(zhuǎn)基因毛白楊的部分個體及枝條木質(zhì)素含量均有所下降,RT-PCR及Real-Time PCR結(jié)果也表明,SbLIM1和SbbHLH1轉(zhuǎn)基因毛白楊中木質(zhì)素生物合成途徑相關(guān)基因的表達(dá)下降的種類和數(shù)量不盡相同。說明這兩個高粱的轉(zhuǎn)錄因子以不同的方式調(diào)控毛白楊木質(zhì)素的合成,該結(jié)果也表明利用SbLIM1和SbbHLH1轉(zhuǎn)基因楊樹,可以獲得低木質(zhì)素的生物纖維材料,有望成為新的高效生物質(zhì)能源。
[Abstract]:Sorghum is one of the important energy crops, its bmr mutant shows low lignin content. Yan Li (2011) used sorghum wild-type BTx623 as tester, and 13 different bmr mutants as driver to construct suppression subtractive (SSH) library. A large number of transcriptional differentially expressed genes were identified by microarray hybridization. SbbHLH1 and SbLIM1 were two distinct transcription factor genes. Previous studies have found that SbbHLH1 and SbLIM1 affect lignin biosynthesis in Arabidopsis thaliana. In this paper, the mechanism of lignin synthesis and metabolism regulated by SbbHLH1 and the genetic transformation of Populus tomentosa were studied, and the effects of these two transcription factors on lignin synthesis were discussed. The interaction protein with SbbHLH1 transcription factors could be studied by yeast double cross technique. The results showed that SbbHLH1 transcription factors could interact with SB02G040140 protein (also a bHLH class protein) in sorghum. These results suggest that SbbHLH1 transcription factors bind to other bHLH domain proteins and participate in lignin transcriptional regulatory networks. DNA motifs combined with SbbHLH1 transcription factors were isolated by SELEX technique. In order to verify that SbbHLH1 transcription factors can bind E-box motifs of Arabidopsis thaliana lignin biosynthesis pathway related gene promoters, DNA motifs were isolated from Arabidopsis thaliana. By cloning these sequences, SbbHLH1 transcription factors can bind to the promoter of AtPAL gene by DNA and protein binding experiments, which further proves that SbbHLH1 transcription factors can regulate lignin biosynthesis by binding PAL gene in Arabidopsis thaliana. Agrobacterium tumefaciens carrying vector pSTART-SbLIM1 was used to infect the explants of Populus tomentosa sprouts. Twelve positive plants were obtained by tissue culture, kanamycin screening and PCR identification. At the same time, the lignin content of three year old SbbHLH1 transgenic Populus tomentosa and SbLIM1 transgenic Populus tomentosa were determined. The results showed that the lignin content of some individuals and branches of transgenic Populus tomentosa decreased, and the results of RT-PCR and Real-Time PCR also showed that the lignin content of some individuals and branches of transgenic Populus tomentosa was decreased. The type and number of genes related to lignin biosynthesis pathway in SbLIM1 and SbbHLH1 transgenic Populus tomentosa were different. The results indicated that the transcription factors of these two sorghum regulated the lignin synthesis in different ways. The results also showed that SbLIM1 and SbbHLH1 transgenic poplars could be used to obtain low lignin bio-fiber materials, which could become new and efficient biomass energy.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2
本文編號：2356625
[Abstract]:Sorghum is one of the important energy crops, its bmr mutant shows low lignin content. Yan Li (2011) used sorghum wild-type BTx623 as tester, and 13 different bmr mutants as driver to construct suppression subtractive (SSH) library. A large number of transcriptional differentially expressed genes were identified by microarray hybridization. SbbHLH1 and SbLIM1 were two distinct transcription factor genes. Previous studies have found that SbbHLH1 and SbLIM1 affect lignin biosynthesis in Arabidopsis thaliana. In this paper, the mechanism of lignin synthesis and metabolism regulated by SbbHLH1 and the genetic transformation of Populus tomentosa were studied, and the effects of these two transcription factors on lignin synthesis were discussed. The interaction protein with SbbHLH1 transcription factors could be studied by yeast double cross technique. The results showed that SbbHLH1 transcription factors could interact with SB02G040140 protein (also a bHLH class protein) in sorghum. These results suggest that SbbHLH1 transcription factors bind to other bHLH domain proteins and participate in lignin transcriptional regulatory networks. DNA motifs combined with SbbHLH1 transcription factors were isolated by SELEX technique. In order to verify that SbbHLH1 transcription factors can bind E-box motifs of Arabidopsis thaliana lignin biosynthesis pathway related gene promoters, DNA motifs were isolated from Arabidopsis thaliana. By cloning these sequences, SbbHLH1 transcription factors can bind to the promoter of AtPAL gene by DNA and protein binding experiments, which further proves that SbbHLH1 transcription factors can regulate lignin biosynthesis by binding PAL gene in Arabidopsis thaliana. Agrobacterium tumefaciens carrying vector pSTART-SbLIM1 was used to infect the explants of Populus tomentosa sprouts. Twelve positive plants were obtained by tissue culture, kanamycin screening and PCR identification. At the same time, the lignin content of three year old SbbHLH1 transgenic Populus tomentosa and SbLIM1 transgenic Populus tomentosa were determined. The results showed that the lignin content of some individuals and branches of transgenic Populus tomentosa decreased, and the results of RT-PCR and Real-Time PCR also showed that the lignin content of some individuals and branches of transgenic Populus tomentosa was decreased. The type and number of genes related to lignin biosynthesis pathway in SbLIM1 and SbbHLH1 transgenic Populus tomentosa were different. The results indicated that the transcription factors of these two sorghum regulated the lignin synthesis in different ways. The results also showed that SbLIM1 and SbbHLH1 transgenic poplars could be used to obtain low lignin bio-fiber materials, which could become new and efficient biomass energy.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2
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