The present invention discloses Litopenaeus vannamei Na, primers and K method ATPase alpha subunit gene and SNP marker screening. According to the invention of Litopenaeus vannamei Na, 12 pairs of primers were designed to complete mRNA sequence K ATPase alpha subunit, amplified by PCR, sequencing and splicing, obtained in Litopenaeus vannamei Na, complete sequence of K gene ATPase alpha subunit. The primers were designed for the sequence segments, and then SNP loci were screened, and 78 SNP loci were obtained. The present invention establishes litopenaeusvannamei Na technology system K ATPase alpha subunit gene SNP marker development, for further screening and resistance of Litopenaeus vannamei SNP markers and related to the development of molecular marker for Litopenaeus vannamei paternity basis, thus speeding up the process of breeding varieties resistance of Litopenaeus vannamei.
【技术实现步骤摘要】
凡纳滨对虾Na,K-ATPaseα亚基基因及SNP标记筛查的扩增引物和方法
:本专利技术涉及水产生物
,具体涉及凡纳滨对虾Na,K-ATPaseα亚基基因及SNP标记筛查的扩增引物和方法。
技术介绍
:凡纳滨对虾(Litopenaeusvannamei)俗称南美白对虾,原产于南美洲,从墨西哥西南沿海至秘鲁西部的太平洋沿岸均有分布,是全世界产量最大的养殖对虾。在我国,凡纳滨对虾养殖区域遍布于全国沿海的海水养殖区和内陆的淡水养殖区,养殖面积约25万公顷,年产量超过100万吨,海水养殖区和淡水养殖区产量各约占50%,年产值300-400亿元人民币,是我国水产养殖的支柱性产业。凡纳滨对虾在海水和淡水中均能成活,这与其强大的渗透压调节能力有着密切的关系,而在凡纳滨对虾的渗透压调节过程中渗透压调节基因起着至关重要的作用。在渗透压调节基因中,Na,K-ATPase基因的作用尤为突出。Na,K-ATPase是有机体赖以生存的重要膜蛋白,参与细胞两侧Na+、K+离子的跨膜主动运输,在维持细胞的膜电位及细胞内外的渗透压平衡,为细胞转运无机离子和营养物质以及排出代谢产物提供驱动力等方面起重要作用。Na,K-ATPase基因是甲壳动物重要的渗透压调节基因,研究显示在低盐度应激后凡纳滨对虾组织中Na,K-ATPase基因的表达量显著上调,表明Na,K-ATPase基因是一个与凡纳滨对虾盐度抗逆相关的功能基因,可望用于凡纳滨对虾耐低盐分子标记的开发,加快凡纳滨对虾耐低盐优良品种的选育进程。在国际上,以分子标记为基础的分子标记辅助选育技术已成为当代水产育种的关键技术。单核苷酸多态 ...
【技术保护点】
一种凡纳滨对虾Na,K‑ATPaseα亚基基因扩增引物,其特征在于,包括12对扩增引物:引物对1:GLvNK‑F1:5’‑CACAGACGGTGAAGAATG‑3’;GLvNK‑R1:5’‑TGGGACCTTGTGCTCATC‑3’;引物对2:GLvNK‑F2:5’‑TGGAACTTGATGAGCACAA‑3’;GLvNK‑R2:5’‑GGCAATGAAGCAGAGGATA‑3’;引物对3:GLvNK‑F3:5’‑CTCCTGCTGTGGATTGGC‑3’;GLvNK‑R3:5’‑ATGAGAAGACGCCTGTGA‑3’;引物对4:GLvNK‑F4:5’‑GGAGCCCAACAAGGACAA‑3’;GLvNK‑R4:5’‑GACAGCATTGGTGGAGAA‑3’;引物对5:GLvNK‑F5:5’‑TTCTCCACCAATGCTGTC‑3’;GLvNK‑R5:5’‑GGCTACAATGATACCAATGAG‑3’;引物对6:GLvNK‑F6:5’‑TGTTGTGTTCCTCATTGGT‑3’;GLvNK‑R6:5’‑CAGTCTCGTGGATGGATAC‑3’;引物对7:GLvNK‑ ...
【技术特征摘要】
1.一种凡纳滨对虾Na,K-ATPaseα亚基基因扩增引物,其特征在于,包括12对扩增引物:引物对1:GLvNK-F1:5’-CACAGACGGTGAAGAATG-3’;GLvNK-R1:5’-TGGGACCTTGTGCTCATC-3’;引物对2:GLvNK-F2:5’-TGGAACTTGATGAGCACAA-3’;GLvNK-R2:5’-GGCAATGAAGCAGAGGATA-3’;引物对3:GLvNK-F3:5’-CTCCTGCTGTGGATTGGC-3’;GLvNK-R3:5’-ATGAGAAGACGCCTGTGA-3’;引物对4:GLvNK-F4:5’-GGAGCCCAACAAGGACAA-3’;GLvNK-R4:5’-GACAGCATTGGTGGAGAA-3’;引物对5:GLvNK-F5:5’-TTCTCCACCAATGCTGTC-3’;GLvNK-R5:5’-GGCTACAATGATACCAATGAG-3’;引物对6:GLvNK-F6:5’-TGTTGTGTTCCTCATTGGT-3’;GLvNK-R6:5’-CAGTCTCGTGGATGGATAC-3’;引物对7:GLvNK-F7:5’-CAAGTATCCATCCACGAGA-3’;GLvNK-R7:5’-AGCCTTTGCTTCAGTGGG-3’;引物对8:GLvNK-F8:5’-ATTCCCATCAAGGAGGTT-3’;GLvNK-R8:5’-GGATGTTAGATGTCAGGGT-3’;引物对9:GLvNK-F9:5’-CCTGACATCTAACATCCCTGAG-3’;GLvNK-R9:5’-GCTTGTCGGTGAATGGGT-3’;引物对10:GLvNK-F10:5’-CCTGCCATTTCCCTTGCC-3’;GLvNK-R10:5’-CGCTCACGGAGACCAAAG-3’;引物对11:GLvNK-F11:5’-ATCATGGCTGAGAACGGCTTCC-3’;GLvNK-R11:5’-TGGAGAACGGAAGAGCAG-3’;引物对12:GLvNK-F12:5’-TTCCGTTCTCCATCCTTA-3’;GLvNK-R12:5’-ATGGAGCAGTAGTTGACC-3’。2.一种凡纳滨对虾Na,K-ATPaseα亚基基因的扩增方法,其特征在于,包括以下步骤:以凡纳滨对虾基因组DNA为模板,分别以权利要求1所述的引物对GLvNK-F1/GLvNK-R1、GLvNK-F2/GLvNK-R2、GLvNK-F3/GLvNK-R3、GLvNK-F4/GLvNK-R4、GLvNK-F5/GLvNK-R5、GLvNK-F6/GLvNK-R6、GLvNK-F7/GLvNK-R7、GLvNK-F8/GLvNK-R8、GLvNK-F9/GLvNK-R9、GLvNK-F10/GLvNK-R10、GLvNK-F11/GLvNK-R11和GLvNK-F12/GLvNK-R12作为扩增引物进行PCR扩增,分别得到扩增产物,然后对扩增产物进行测序,然后再进行序列拼接,获得凡纳滨对虾Na,K-ATPaseα亚基的完整基因序列。3.根据权利要求2所述的扩增方法,其特征在于,所述的PCR扩增,其反应体系为25μL,包括:不含Mg2+的10×PCRbuffer2.5μL、25mMMgCl22.0μL,10mMdNTP0.5μL、5U/μLLATaq酶0.2μL、10μM正向引物0.5μL、10μM反向引物0.5μL、DNA模板12.5ng,其余由无菌水补足至25μL。4.根据权利要求2或3所述的扩增方法,其特征在于,所述的PCR扩增,其反应程序为:95℃预变性3分钟;95℃变性30秒,55℃退火30秒,72℃延伸2分钟,共35个循环;72℃再延伸10分钟。5.一种凡纳滨对虾Na,K-ATPaseα亚基基因SNP标记筛查的扩增引物,其特征在于,包括13对扩增引物:引物对13:LvNK001-LvNK011-F:5’-TGGAACTTGATGAGCACAA-3’;LvNK001-LvNK011-R:5’-GGCAATGAAGCAGAGGATA-3’;引物对14:LvNK012-LvNK023-F:5’-CTCCTGCTGTGGATTGGC-3’;LvNK012-LvNK023-R:5’-GACTTTGCCTCCTGATAG-3’;引物对15:LvNK024-LvNK027-F:5’-GGCGTCTTCTCCTACTATCA-3’;LvNK024-LvNK027-R:5’-AAAGCAAAGGGAAAGTGA-3’;引物对16:LvNK028-LvNK030-F:5’-GCAGTTGTACCTGGGCATTG-3’;LvNK028-LvNK030-R:5’-GACAGCATTGGTGGAGAA-3’;引物对17:LvNK031-LvNK036-F:5’-TTCTCCACCAATGCTGTC-3’;LvNK031-LvNK036-R:5’-GGCTACAATGATACCAATGAG-3’;引物对18:LvNK037-LvNK039-F:5’-TGTTGTGTTCCTCATTGGT-3’;LvNK037-LvNK039-R:5’-AGGTAGGCATTGTTGAAAG-3’;引物对19:LvNK040-LvNK042-F:5’-ATTCCCATCAAGGAGGTT-3’;LvNK040-LvNK042-R:5’-GGATGTTAGATGTCAGGGT-3’;引物对20:LvNK043-LvNK046-F:5’-CATTGCTTACACCCTGAC-3’;LvNK043-LvNK046-R:5’-CAGGGAATGAACTTGTAGG-3’;引物对21:LvNK047-LvNK052-F:5’-TATTTAGGTGCCT...
【专利技术属性】
技术研发人员:任春华,江晓,陈廷,黄文,胡超群,
申请(专利权)人:中国科学院南海海洋研究所,
类型:发明
国别省市:广东,44
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