紫花苜蓿MsCOL1基因RNAi表达载体的构建及转化
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摘要
根据紫花苜蓿CONSTANS类似基因MsCOL1基因(登录号:DQ661682)序列,设计含有酶切位点的两对特异性引物,以紫花苜蓿cDNA为模板,分别合成用于构建干扰载体的正反义片段,将正反义片段分别插入表达载体pART27的相应位置,构建含有发夹结构的RNAi载体pART-S-A。利用农杆菌介导方法,将pART-S-A转化到紫花苜蓿中,经PCR检测,获得了7株转基因植株。经RT-PCR检测,证明转基因植株中MsCOL1基因表达量有所下降,其中5株的表达量明显的降低。结果表明,已构建成功具有发夹结构的RNAi载体pART-S-A,它可有效的抑制紫花苜蓿MsCOL1基因。
Based on the sequence of alfalfa Constans-Like 1(MsCOL1) gene(Access No.: DQ661682),two pairs of specific primers containing different enzyme sites were designed.With the template of full-length cDNA,positive-sense strand and antisense strand were obtained,which were separately inserted into the restrict sites of expression vector pART27.The RNAi vector of pART-S-A containing a hairpin structure was constructed.Mediated by Agrobacterium tumefaciens,pART-S-A was transformed into alfalfa plants.PCR testing showed that seven transgenic plants were obtained.The result of RT-PCR showed that transgenic alfalfa had lower expression level of MsCOL1 and five of those exhibited obviously low expression levels of MsCOL1.These results indicated that the RNAi vector pART-S-A containing a hairpin structure was constructed successfully and the alfalfa MsCOL1 gene was decreased effectively.
Based on the sequence of alfalfa Constans-Like 1(MsCOL1) gene(Access No.: DQ661682),two pairs of specific primers containing different enzyme sites were designed.With the template of full-length cDNA,positive-sense strand and antisense strand were obtained,which were separately inserted into the restrict sites of expression vector pART27.The RNAi vector of pART-S-A containing a hairpin structure was constructed.Mediated by Agrobacterium tumefaciens,pART-S-A was transformed into alfalfa plants.PCR testing showed that seven transgenic plants were obtained.The result of RT-PCR showed that transgenic alfalfa had lower expression level of MsCOL1 and five of those exhibited obviously low expression levels of MsCOL1.These results indicated that the RNAi vector pART-S-A containing a hairpin structure was constructed successfully and the alfalfa MsCOL1 gene was decreased effectively.
引文
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[2]Robson F,Costa MMR,Hepworth SR,et al.Functional im-portance of conserved domains in the flowering-time geneCONSTANSdemonstrated by analysis of mutant alleles and trans-genic plants[J].The Plant Journal,2001,28(6):619-631.
[3]Paula Suárez-López,Kay Wheatley,Frances Robson,et al.CONSTANS mediates between the circadian clock and thecontrol of flowering in Arabidopsis[J].Nature,2001,410(6832):1116-1120.
[4]Igor Kardailsky,Vipula K Shukla,Ji Hoon Ahn,et al.Acti-vation tagging of the floral inducer FT[J].Science,1999,286(5446):1962-1965.
[5]Hitoshi Onouchi,M.Isabel Igeno,Claire Périlleux,et al.Mutagenesis of plants overexpressing CONSTANS demon-strates novel interactions among Arabidopsis flowering-timegenes[J].Plant Cell,2000,12(6):885-900.
[6]Alon Samach,Hitoshi Onouchi,Scott E Gold,et al.Distinctroles of CONSTANStarget genes in reproductive developmentof Arabidopsis[J].Science,2000,288(5471):1613-1616.
[7]Horim Lee,Sung-Suk Suh,Eunsook Park.The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductivepathways in Arabidopsis[J].Genes Dev,2000,14(18):2366-2376.
[8]Shiv B Tiwari,Yu Shen,Han-chang Chang,et al.The flower-ing time regulator CONSTANSis recruited to the FLOWER-ING LOCUS T promoter via a unique cis-element[J].NewPhytol,2010,187(1):57-66.
[9]Susan Ledger,Carl Strayer,Fern Ashton,et al.Analysis ofthe function of two circadian-regulated CONSTANS-LIKEgenes[J].Plant J.,2001,26(1):15-22.
[10]Xiao fei Cheng,Zengyu Wang.Overexpression of COL9,aCONSTANS-LIKE gene,delays flowering by reducing ex-pression of CO and FT in Arabidopsis thaliana[J].PlantJ.,2005,43(5):758-768.
[11]N D González-Schain,P Suárez-López.CONSTANSdelaysflowering and affects tuber yield in potato[J].Biologia Plan-tarum,2008,52(2):251-258.
[12]Sourav Datta,G H C M Hettiarachchi,Xingwang Deng,etal.Arabidopsis CONSTANS-LIKE3is a positive regulator ofred light signaling and root growth[J].The Plant Cell On-line,2006,18(1):70-84.
[13]Soon Kap Kim,ChoongHyo Yun,Jeong Hwan Lee,et al.OsCO3,a CONSTANS-LIKE gene,controls flowering bynegatively regulating the expression of FT-like genes underSD conditions in rice[J].Planta,2008,228(2):355-365.
[14]关淑艳,楚海娇,刘慧婧,等.玉米sbe2a基因RNAi载体的构建及转化玉米的初步研究[J].吉林农业大学学报,2011,33(2):172-176.Guan Shuyan,Chu Haijiao,Liu Huijing,et al.The primarystudy on construction of RNAi expression vector of sbe2agene and maize transformation[J].Journal of Jilin Agri-cultural University,2011,33(2):172-176.
[15]秦智慧,晁跃辉,杨青川,等.紫花苜蓿锌指蛋白基因RNAi表达载体的构建及在苜蓿的转化[J].作物学报,2010,36(4):596-601.Qin Zhihui,Chao Yuehui,Yang Qingchuan,et al.Construc-tion and transformation of RNAi vector of MsZFN Gene fromalfalfa(Medicago sativa L.)[J].acta Agronomica Sinica,2010,36(4):596-601.
[16]张付云,李妍,李云冰,等.NtSKP1基因的RNAi载体构建及对烟草的转化[J].西北农业学报,2010,19(8):95-100.Zhang Fuyun,Li Yan,Li Yunbing,et al.Construction ofRNAi expression vector of NtSKP1and transformation in to-bacco[J].Acta Agriculturae Boreali-occidentalis Sinica,2010,19(8):95-100.
[17]G Hutvágner,L Mlynárová,and J P Nap.Detailed charac-terization of the posttranscriptional gene-silencing-relatedsmall RNA in a GUS gene-silenced tobacco[J].RNA,2000,6(10):1445-1454.