柿果实CCD1基因超表达和RNAi表达载体的构建
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摘要
采用CTAB法提取小方柿果肉总RNA,根据带有不同酶切位点的引物获得目的基因柿类胡萝卜素裂解双加氧酶基因(DkCCD1),将扩增的目的基因序列与NCBI登录的基因序列进行核苷酸序列和氨基酸序列比对,发现核苷酸序列相似度均超过99%,氨基酸序列相似度均为100%,由此得到可用于后续试验的目的基因。将得到的目的基因与双元表达载体pCAMBIA1301连接,构建pCAMBIA1301-CCD1超表达载体和具有发卡结构的pCAMBIA1301-CCD1-RNAi表达载体,通过冻融法将载体导入农杆菌EHA105。PCR扩增结果表明:所构建的pCAMBIA1301-CCD1超表达载体和pCAMBIA1301-CCD1-RNAi载体已导入农杆菌,可用于后续的遗传转化研究。
Total RNA was extracted from fruit pulp of persimmon‘Xiaofangshi'with CTAB method.The target gene carotenoid cleavage dioxigenase 1 gene(DkCCD1)was amplified by primers with different restriction enzyme sites.The results of nucleotide sequence and amino acid sequence alignments showed that the similarity of the nucleotide sequence was more than 99% and the similarity of the amino acid sequence was 100%,which indicated that the sequence can be used in the following experiment.The target gene was ligated with the binary expression vector pCAMBIA1301.The over-expression vector pCAMBIA1301-CCD1 and the RNAi expression vector pCAMBIA1301-CCD1-RNAi with hairpin structure were successfully constructed,and then transferred to Agrobacterium EHA105 by freeze-thawing method.PCR amplification results showed that the pCAMBIA1301-CCD1 overexpression vector and pCAMBIA1301-CCD1-RNAi vectors were introduced into Agrobacterium,which could be used for subsequent genetic transformation research.
Total RNA was extracted from fruit pulp of persimmon‘Xiaofangshi'with CTAB method.The target gene carotenoid cleavage dioxigenase 1 gene(DkCCD1)was amplified by primers with different restriction enzyme sites.The results of nucleotide sequence and amino acid sequence alignments showed that the similarity of the nucleotide sequence was more than 99% and the similarity of the amino acid sequence was 100%,which indicated that the sequence can be used in the following experiment.The target gene was ligated with the binary expression vector pCAMBIA1301.The over-expression vector pCAMBIA1301-CCD1 and the RNAi expression vector pCAMBIA1301-CCD1-RNAi with hairpin structure were successfully constructed,and then transferred to Agrobacterium EHA105 by freeze-thawing method.PCR amplification results showed that the pCAMBIA1301-CCD1 overexpression vector and pCAMBIA1301-CCD1-RNAi vectors were introduced into Agrobacterium,which could be used for subsequent genetic transformation research.
引文
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[2]WINTERHALTER P,SCHREIER P.C13-norisoprenoid glycosides in plant tissues:an overview on their occurrence,composition and role as flavour precursors[J].Flavour and Fragrance Journal,1994,9:281-287.
[3]LEWINSOHN E,YARON S,EINAT B,et al.Not just colors-carotenoid degradation as a link between pigmentation and aroma in tomato and watermelon fruit[J].Trends in Food Science&Technology,2005,16:407-415.
[4]MICHELE E,BLOCK A A,JONATHAN T V,et al.Characterization of three members of the Arabidopsis carotenoidcleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme family[J].Plant Journal,2006,45(6):982-993.
[5]温可睿.葡萄类胡萝卜素裂解双加氧酶基因克隆及功能鉴定[D].哈尔滨:东北林业大学,2012.
[6]张风杰.柿果实香气分析及类胡萝卜素裂解双加氧酶(CCDs)基因克隆与表达研究[D].扬州:扬州大学,2013.
[7]卜庆忠.柿果实外观品质指标及香气成分检测研究[D].扬州:扬州大学,2016.
[8]杨跃飞,祁钰钰,杨开典,等.小鼠MSTN shRNA可控表达载体构建及基因沉默效率研究[J].扬州大学学报(农业与生命科学版),2017,37(4):1-4.
[9]魏恬恬,臧姝,曾雨倩,等.植物单萜类化合物的代谢调控[J].扬州大学学报(农业与生命科学版),2017,38(2):122-126.
[10]毕惠惠,王根平,王成社,等.单子叶植物RNA干扰和过表达Gateway载体的构建[J].植物遗传资源学报,2013,14(1):115-123.
[11]FIRE A,XU S,MONTGOMERY M K,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391:806-811.
[12]YOUNIS A,SIDDIQUE M I,KIM C K,et al.RNA interference(RNAi)induced gene silencing:apromising approach of Hi-tech plant breeding[J].International Journal of Biological Sciences,2014,10(10):1150-1158.
[13]朱龙付,张献龙.RNAi及其在植物遗传改良中的应用[J].华中农业大学学报,2004,23(4):472-477.
[14]徐昌杰,陈昆松,张波,等.柑橘组织RNA提取方法研究[J].果树学报,2004,21(2):136-140.
[15]JAGTAP U B,GURAV R G,BAPAT V A.Role of RNA interference in plant improvement[J].Naturwissens Chaften,2011,98:473-492.
[16]张恭,刘立峰,马峙英.RNA干扰及其植物抗病毒应用[J].中国农学通报,2007,23(1):42-45.
[17]SMITH N A,SINGH S P,WANG M B,et al.Total silencing by intron spliced hairpin RNAs[J].Nature,2000,407:319-320.
[18]李丽文,朱延明,李杰,等.RNAi技术在植物功能基因组学中的研究进展[J].东北农业大学学报,2007,38(1):119-124.