魔芋GDP-甘露糖焦磷酸化酶基因及其启动子的克隆与分析
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摘要
以花魔芋和白魔芋为材料,通过魔芋5个转录组高通量测序数据库,与拟南芥进行tBlastn比对、拼接,得到魔芋中的GDP-甘露糖焦磷酸化酶基因GMP的推定全长,设计基因特异性引物,克隆得到魔芋GMP基因的部分cDNA序列,长度为1 233 bp,其中基因编码区长度为1 086 bp,编码的氨基酸为361个.在此基础上运用FNPI-PCR技术得到了魔芋GDP-甘露糖焦磷酸化酶基因GMP上游启动子2 116 bp,经生物信息学分析,该序列具有典型的TATA-box、 CAAT-box及与胁迫相关的元件和光应答元件.
In this study, tBlastn searches of high-throughput transcriptome sequencing databases of Amorphophallus konjac and A. albus were aligned and spliced with the amino acid sequence of Arabidopsis thaliana, and the GMP transcript was assembled. The putative full-length sequence of the GMP gene was cloned. By designing gene-specific primers, a partial cDNA sequence of konjac GMP gene with a length of 1 233 bp was cloned. The length of the coding region of the gene was 1086 bp, and the encoded amino acid was 361. Based on the above results, using FNPI-PCR technology, we got konjac GDP-mannose pyrophosphorylase gene GMP upstream promoter 2 116 bp in length. According to bioinformatics analysis, typical TATA-box, CAAT-box, and stress-related elements and light responsive elements and other cis-elements were identified in the promoter region.
In this study, tBlastn searches of high-throughput transcriptome sequencing databases of Amorphophallus konjac and A. albus were aligned and spliced with the amino acid sequence of Arabidopsis thaliana, and the GMP transcript was assembled. The putative full-length sequence of the GMP gene was cloned. By designing gene-specific primers, a partial cDNA sequence of konjac GMP gene with a length of 1 233 bp was cloned. The length of the coding region of the gene was 1086 bp, and the encoded amino acid was 361. Based on the above results, using FNPI-PCR technology, we got konjac GDP-mannose pyrophosphorylase gene GMP upstream promoter 2 116 bp in length. According to bioinformatics analysis, typical TATA-box, CAAT-box, and stress-related elements and light responsive elements and other cis-elements were identified in the promoter region.
引文
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[5]GILLE S,CHENG K,SKINNER ME,et al.Deep Sequencing of Voodoo Lily(Amorphophallus Konjac)an Approach to Identify[J].Planta,2011,234(3):515-526.
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[7]郑岑,张立平,唐忠辉,等.TAIL-PCR技术及其在植物基因中的克隆[J].基因组学和应用生物学,2009,28(3):544-548.
[8]程国灵,许文涛,石慧,等.A-T连接接头PCR基因组步移技术[J].农业生物技术学报,2013,21(12):1527-1527.
[9]李婧,曾媛,龚胜,等.大花三色堇FPNI-PCR反应体系的优化[J].江苏农业科学,2016,44(5):72-75.
[10]刘祥,赵志新,王斐,等.蜡梅水通道蛋白基因CpAQP1转录水平变化及其启动子克隆[J].西南大学学报(自然科学版),2014,36(2):48-55.
[11]SAWAKE S,TAJIMA N,MORTIMER JC,et al.KONJAC1and 2are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis[J].Plant Cell,2015,27(12):3397-3409.
[12]邵铁梅,仵陶,焦展,等.油葵种子特异性基因HaAP10启动子克隆及功能验证[J].西南师范大学学报(自然科学版),2016,41(10):39-45.
[2]HE C M,ZENG S J,da SILVA J A T,et al.Molecular Cloning and Functional Analysis of the Phosphomannomutase(PMM)Gene fromDendrobium Officinale and Evidence for the Involvement of an Abiotic Stress Response during Germination[J].Protoplasma,2017,254(4):1693-1704.
[3]HE C M,YU Z M,da SILVA J A T,et al.DoGMP1from Dendrobium Officinale Contributes to Mannose Content of WaterSoluble Polysaccharides and Plays a Role in Salt Stress Response[J].Scientific Reports,2017,7:41010-1-41010-13.
[4]BARTH C,GOUZD ZA,STEELE HP,et al.A Mutation in GDP-Mannose PyrophosphorylaseCauses Conditional Hypersensitivity to Ammonium,Resulting in Arabidopsis Root Growth Inhibition,Altered Ammonium Metabolism,and Hormone Homeostasis[J].Journal of Experimental Botany,2010,61(2):379-394.
[5]GILLE S,CHENG K,SKINNER ME,et al.Deep Sequencing of Voodoo Lily(Amorphophallus Konjac)an Approach to Identify[J].Planta,2011,234(3):515-526.
[6]WANG Z,YE S F,LI J J,et al.Fusion Primer and Nested Integrated PCR(FPNI-PCR):A New High-Efficiency Strategy for Rapid Chromosome Walking or Flanking Sequence Cloning[J].BMC Biotechnology,2011,11:109-121.
[7]郑岑,张立平,唐忠辉,等.TAIL-PCR技术及其在植物基因中的克隆[J].基因组学和应用生物学,2009,28(3):544-548.
[8]程国灵,许文涛,石慧,等.A-T连接接头PCR基因组步移技术[J].农业生物技术学报,2013,21(12):1527-1527.
[9]李婧,曾媛,龚胜,等.大花三色堇FPNI-PCR反应体系的优化[J].江苏农业科学,2016,44(5):72-75.
[10]刘祥,赵志新,王斐,等.蜡梅水通道蛋白基因CpAQP1转录水平变化及其启动子克隆[J].西南大学学报(自然科学版),2014,36(2):48-55.
[11]SAWAKE S,TAJIMA N,MORTIMER JC,et al.KONJAC1and 2are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis[J].Plant Cell,2015,27(12):3397-3409.
[12]邵铁梅,仵陶,焦展,等.油葵种子特异性基因HaAP10启动子克隆及功能验证[J].西南师范大学学报(自然科学版),2016,41(10):39-45.