强旱生植物沙冬青AmDHN、AmERF基因克隆及转化甜菜的研究
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摘要
甜菜是世界上重要的糖料作物之一,也是我国第二大糖料作物,在我国国民生产总值中占有重要地位。本试验是在已对强旱生植物沙冬青cDNA文库研究的基础上,通过EST序列分析和GENBANK核酸数据库的比对,对检测到的34个DHN基因ESTs和15个ERF的ESTs进行拼接得到基因全长的cDNA序列,通过设计特异性引物克隆了沙冬青AmDHN基因和AmERF转录因子,构建了沙冬青AmDHN基因植物表达载体和沙冬青AmDHN基因、AmERF转录因子及PPT抗性基因二价植物表达载体,建立了甜菜组培再生体系,利用农杆菌介导法将沙冬青AmDHN基因导入甜菜,并进行了功能验证。主要研究结果入如下:
1.克隆了沙冬青脱水素(AmDHN)基因,其全长为1106bp,读码框为552bp,编码183个氨基酸,理论MW=18407.4,理论pI=6.22。
2.克隆了沙冬青乙烯应答转录因子(AmERF),其读码框为633bp,编码211个氨基酸,理论MW=23216.0,理论pI=8.20。网络Blastx程序比对,找到56个同源性较高的序列,其中与拟南芥(Arabidopsis thaliana)、水稻粳稻品种组(japonica cultivar-group of Oryza sativa)、辣椒(Capsicum annuum)、蒺藜苜蓿(Medicago truncatula)的一致性分别达到83%、72%、74%和61%。
3.利用PCR定点突变技术对AmDHN基因的1个碱基进行了定点突变,成功地将其碱基序列CCATGG突变为CTATGG。
4.构建了沙冬青的AmDHN基因植物表达载体pCAMBIA3301-DHN与AmDHN基因、AmERF转录因子的二价植物表达载体pCAMBIA3301-DHN-AmERF,为今后进一步研究沙冬青抗逆基因的表达规律及代谢调控等提供了基础,同时也丰富了利用转基因技术提高作物抗逆性的基因库。
5.建立了以甜菜叶柄为外植体的遗传转化再生体系,筛选出诱导丛生芽能力较高的甜菜基因型材料4个(N98122、N9849、HBB-1和内甜单1),分化诱导培养基2种(MS+NAA0.5mg/L+6-BA1.5mg/L;MS+NAA0.5mg/L+KT3.0mg/L),生根诱导培养基1种(MS+NAA2.0mg/L)。
6.采用农杆菌介导法并结合真空辅助侵染将脱水素基因导入了甜菜,获得经PCR检测阳性转基因植株107株,PPT抗性检测结果显示转基因植株具有很强的PPT抗性,干旱胁迫试验结果显示转基因甜菜植株的抗旱性要高于非转基因甜菜植株。
Sugar beet is one of important sugar crops in the world and the second largest sugar crop in China. It plays an important role in the development of sugar beet industry. A cDNA library of strong xerophyte Ammopiptanthus has been known. We analyzed its EST sequence and compared GENBANK DNA database, and then spliced ESTs of 34 DHN genes and 15 ERF genes. A full-length cDNA sequences was got. AmDHN gene and AmERF transcription factor of Ammopiptanthus were cloned using a specific primer. And then, AmDHN gene plant expression vector, AmDHN gene, AmERF transcription factor and PPT screening for resistance gene divalent expression vector of the plant of Ammopiptanthus were built in turn. Sugar beet regeneration tissue culture system was established. Ammopiptanthus AmDHN gene was transferred into sugar beet using Agrobacterium method. Functions of transgenic sugar beet were verified. The results are as follows:
1. AmDHN full length with 1106 bp was successfully cloned by PCR amplification technique. DNAman analysis indicated open read frame was 552 bp, coded 183 aa, theory MW was 18407.4 and pI was 6.22.
2. Transcriptional factor AmERF of Ammopiptanthus was cloned successfully. Open read frame was 633 bp, coded 211 aa, theory MW was 23216.0 and pIwas 8.20 by DNAm- an analysis. Nucleotide sequence of transcriptional factor AmERF of Ammopiptanthus was compared by Blastx procedure, and 56 homologous sequences were founded. Consistency with Arabidopsis thaliana, Oryza sativa (japonica cultivar-group), Capsicum annuum, Medicago truncatula was 83%、72%、74% and 61% respectively.
3. Mutation of base sequence CCATGG of DHN gene to CTATGG was completed successfully using PCR site-directed mutagenesis technique.
4. AmDHN gene plant expression vector (pCAMBIA3301-DHN), DHN gene and divalent expression vector of AmERF transcription factor (pCAMBIA3301-DHN-AmERF) of Ammopiptanthus were built.
5. A sugar beet genetic transformation and regeneration system was built, in which explants were petiole of sugar beet. 4 genotypes sugar beets (N98122、N9849、HBB-1and NeiTianDan1) which have higher ability to induce multiple shoots were screened, compan- ied with 2 differentiation inducing medium (MS+NAA0.5mg/L+6-BA1.5mg/L;MS+NAA 0.5mg/L+KT3.0mg/L) and 1 rooting inducing medium (MS+NAA2.0mg/L).
6. Ammopiptanthus AmDHN gene was transferred into sugar beet integrated Agrobacterium tumefaciens with vacuum, and then got 107 transgenic plants which have stronger PPT resistance. Under drought stress, transgenic sugar beet plant has stronger resistance than non-transgenic ones.
1.克隆了沙冬青脱水素(AmDHN)基因,其全长为1106bp,读码框为552bp,编码183个氨基酸,理论MW=18407.4,理论pI=6.22。
2.克隆了沙冬青乙烯应答转录因子(AmERF),其读码框为633bp,编码211个氨基酸,理论MW=23216.0,理论pI=8.20。网络Blastx程序比对,找到56个同源性较高的序列,其中与拟南芥(Arabidopsis thaliana)、水稻粳稻品种组(japonica cultivar-group of Oryza sativa)、辣椒(Capsicum annuum)、蒺藜苜蓿(Medicago truncatula)的一致性分别达到83%、72%、74%和61%。
3.利用PCR定点突变技术对AmDHN基因的1个碱基进行了定点突变,成功地将其碱基序列CCATGG突变为CTATGG。
4.构建了沙冬青的AmDHN基因植物表达载体pCAMBIA3301-DHN与AmDHN基因、AmERF转录因子的二价植物表达载体pCAMBIA3301-DHN-AmERF,为今后进一步研究沙冬青抗逆基因的表达规律及代谢调控等提供了基础,同时也丰富了利用转基因技术提高作物抗逆性的基因库。
5.建立了以甜菜叶柄为外植体的遗传转化再生体系,筛选出诱导丛生芽能力较高的甜菜基因型材料4个(N98122、N9849、HBB-1和内甜单1),分化诱导培养基2种(MS+NAA0.5mg/L+6-BA1.5mg/L;MS+NAA0.5mg/L+KT3.0mg/L),生根诱导培养基1种(MS+NAA2.0mg/L)。
6.采用农杆菌介导法并结合真空辅助侵染将脱水素基因导入了甜菜,获得经PCR检测阳性转基因植株107株,PPT抗性检测结果显示转基因植株具有很强的PPT抗性,干旱胁迫试验结果显示转基因甜菜植株的抗旱性要高于非转基因甜菜植株。
Sugar beet is one of important sugar crops in the world and the second largest sugar crop in China. It plays an important role in the development of sugar beet industry. A cDNA library of strong xerophyte Ammopiptanthus has been known. We analyzed its EST sequence and compared GENBANK DNA database, and then spliced ESTs of 34 DHN genes and 15 ERF genes. A full-length cDNA sequences was got. AmDHN gene and AmERF transcription factor of Ammopiptanthus were cloned using a specific primer. And then, AmDHN gene plant expression vector, AmDHN gene, AmERF transcription factor and PPT screening for resistance gene divalent expression vector of the plant of Ammopiptanthus were built in turn. Sugar beet regeneration tissue culture system was established. Ammopiptanthus AmDHN gene was transferred into sugar beet using Agrobacterium method. Functions of transgenic sugar beet were verified. The results are as follows:
1. AmDHN full length with 1106 bp was successfully cloned by PCR amplification technique. DNAman analysis indicated open read frame was 552 bp, coded 183 aa, theory MW was 18407.4 and pI was 6.22.
2. Transcriptional factor AmERF of Ammopiptanthus was cloned successfully. Open read frame was 633 bp, coded 211 aa, theory MW was 23216.0 and pIwas 8.20 by DNAm- an analysis. Nucleotide sequence of transcriptional factor AmERF of Ammopiptanthus was compared by Blastx procedure, and 56 homologous sequences were founded. Consistency with Arabidopsis thaliana, Oryza sativa (japonica cultivar-group), Capsicum annuum, Medicago truncatula was 83%、72%、74% and 61% respectively.
3. Mutation of base sequence CCATGG of DHN gene to CTATGG was completed successfully using PCR site-directed mutagenesis technique.
4. AmDHN gene plant expression vector (pCAMBIA3301-DHN), DHN gene and divalent expression vector of AmERF transcription factor (pCAMBIA3301-DHN-AmERF) of Ammopiptanthus were built.
5. A sugar beet genetic transformation and regeneration system was built, in which explants were petiole of sugar beet. 4 genotypes sugar beets (N98122、N9849、HBB-1and NeiTianDan1) which have higher ability to induce multiple shoots were screened, compan- ied with 2 differentiation inducing medium (MS+NAA0.5mg/L+6-BA1.5mg/L;MS+NAA 0.5mg/L+KT3.0mg/L) and 1 rooting inducing medium (MS+NAA2.0mg/L).
6. Ammopiptanthus AmDHN gene was transferred into sugar beet integrated Agrobacterium tumefaciens with vacuum, and then got 107 transgenic plants which have stronger PPT resistance. Under drought stress, transgenic sugar beet plant has stronger resistance than non-transgenic ones.
引文
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