高粱SbbHLH1互作蛋白、DNA结合序列的识别及毛白杨转基因的初步研究
发布时间：2018-11-25 15:38
【摘要】：高粱是重要的能源作物之一,其bmr突变体表现出低木质素含量。闫丽(2011)等利用以高粱野生型BTx623作为tester、以13种不同的bmr突变体作为driver构建的抑制差减杂交(SSH)文库,通过芯片杂交识别出大量转录差异的基因。SbbHLH1和SbLIM1是其中两个差异较明显的转录因子基因。前期的研究发现,SbbHLH1和SbLIM1影响拟南芥的木质素生物合成。本文对SbbHLH1调控木质素合成代谢机制及毛白杨的遗传转化进行初步的研究,探讨这两个转录因子对其木质素合成的影响。采用酵母双杂技术可以研究与SbbHLH1转录因子的互作蛋白,结果发现SbbHLH1转录因子可以与高粱中的SB02G040140蛋白(也是一个bHLH类的蛋白)互作。表明了SbbHLH1转录因子会与其他的bHLH结构域蛋白结合,再参与到木质素的转录调控网络。采用SELEX技术分离得到了与SbbHLH1转录因子结合的DNA基序E-box (CANNTG),为了验证SbbHLH1转录因子能够结合拟南芥木质素生物合成途径相关基因启动子序列中的E-box基序,克隆这些序列,通过DNA和蛋白的结合实验发现SbbHLH1转录因子可以与AtPAL基因的启动子结合,这就进一步证明了SbbHLH1转录因子在拟南芥中可以通过结合PAL基因调控木质素的生物合成。用携带载体pSTART-SbLIM1的农杆菌侵染毛白杨芽尖的外植体,通过组织培养、卡那霉素筛选、PCR鉴定获得12株阳性植株。同时对三年生SbbHLH1转基因毛白杨及SbLIM1转基因毛白杨的木质素含量进行测定,结果发现转基因毛白杨的部分个体及枝条木质素含量均有所下降,RT-PCR及Real-Time PCR结果也表明,SbLIM1和SbbHLH1转基因毛白杨中木质素生物合成途径相关基因的表达下降的种类和数量不尽相同。说明这两个高粱的转录因子以不同的方式调控毛白杨木质素的合成,该结果也表明利用SbLIM1和SbbHLH1转基因杨树,可以获得低木质素的生物纤维材料,有望成为新的高效生物质能源。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
本文编号：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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
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