二倍体马铃薯蛋白酶抑制剂II基因的克隆、序列分析及表达载体的构建

遗传 ›› 2005, Vol. 27 ›› Issue (3): 417-422.

二倍体马铃薯蛋白酶抑制剂II基因的克隆、序列分析及表达载体的构建

卜庆云;武亮;杨世湖;万建民

南京农业大学作物遗传与种质创新国家重点实验室, 江苏省植物基因工程研究中心，南京 210095

收稿日期:2004-04-30 修回日期:2004-05-20 出版日期:2005-06-10 发布日期:2005-06-10
通讯作者: 杨世湖

Cloning, Characterization and Expression Vector Construction of Potato Protease-inhibitor II Gene(PINII-2x) from Diploid Potato （Solanum phurejia）

BU Qing-Yun;WU Liang;YANG Shi-Hu;WAN Jian-Min

National Key Laboratory of Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Center, Nanjing Agricultural University, Nanjing 210095,China

Received:2004-04-30 Revised:2004-05-20 Online:2005-06-10 Published:2005-06-10
Contact: YANG Shi-Hu

摘要/Abstract

摘要： 根据GenBank发表的四倍体马铃薯栽培种(Solanum.tuberosum)来源的马铃薯蛋白酶抑制剂II基因序列，用PCR方法从二倍体马铃薯IVP101（Solanum.phurejia）cDNA文库和基因组DNA扩增得到马铃薯蛋白酶抑制剂II的cDNA和DNA，命名为PINII-2x。测序表明，PINII-2x基因组DNA全长580 bp,含有一个115 bp的内含子，cDNA有462 bp(除去终止密码子)。推测的PINII-2x分子量是16.6 kD,等电点6.08。与其他马铃薯蛋白抑制剂II同源性比较表明，核苷酸同源性高达88%;推测的氨基酸序列同源性为93%,并具有相同的活性中心。RT-PCR表明，PINII-2x的mRNA在叶片中具有强烈的伤害诱导表达特性。同时，构建了分别以水稻ActI启动子和玉米Ubi启动子驱动PINII-2x基因的双元载体。

关键词: 二倍体马铃薯（Solanum.phurejia）, 蛋白酶抑制剂 II, 伤害诱导

Abstract: Based on the published gene sequence of tetraploid potato (Solanum.tuberosum) protease-inhibitor II, a genomic DNA and a cDNA sequence of potato protease-inhibitor II gene were obtained from the cDNA library and the genomic DNA of a diploid potato IVP101 (Solanum.phurejia) using PCR method and named PINII-2x. Nucleotide sequencing confirmed that the full-length DNA of PINII-2x was 580 bp, including an 115 bp intron and two exons. cDNA was 462 bp ( stop codon TGA not included) and had 88% similarity to the tetraploid potato protease-inhibitor II. The PINII-2x open reading frame encodes a 154-amino acid polypeptide with a predicated size of 16.6 KD and a calculated PI of 6.08. The deduced proteins from PINII-2x cDNA had 93% homology with other tetraploid potato protease-inhibitor II, which contain the intact signal peptide and two active site similar to the potato protease-inhibitor II family. Test of the RT-PCR indicated that PINII-2x mRNA is wound- induced expression in potato leaves. Binary vector of PINII-2x cDNA drove by either rice Actin I promoter (ActI) or maize Ubiquitin promoter (Ubi) was constructed.

中图分类号:

Q943

卜庆云，武亮，杨世湖，万建民. 二倍体马铃薯蛋白酶抑制剂II基因的克隆、序列分析及表达载体的构建[J]. 遗传, 2005, 27(3): 417-422.

BU Qing-Yun, WU Liang, YANG Shi-Hu, WAN Jian-Min. Cloning, Characterization and Expression Vector Construction of Potato Protease-inhibitor II Gene(PINII-2x) from Diploid Potato （Solanum phurejia）[J]. HEREDITAS, 2005, 27(3): 417-422.

[1]	田云，卢向阳，彭丽莎，方俊. 植物WRKY转录因子结构特点及其生物学功能[J]. 遗传, 2006, 28(12): 1607-1607~1612.
[2]	刘成，杨足君，冯娟，周建平，迟世华，任正隆. 利用小麦微卫星引物建立簇毛麦染色体组特异性标记[J]. 遗传, 2006, 28(12): 1573-1573~1579.
[3]	陈晓军，叶春江，吕慧颖，徐民新，李葳，张利明，王超，罗淑萍，朱保葛. GmHSFA1基因克隆及其过量表达提高转基因大豆的耐热性[J]. 遗传, 2006, 28(11): 1411-1420.
[4]	黄艳梅，祁英杰，徐基清，朱运良，伍新尧. 2个新Y-STR基因座的序列分析和在广东汉族群体中的多态性研究[J]. 遗传, 2006, 28(11): 1355-1360.
[5]	秦君，姜成喜，刘章雄，付亚书，关荣霞，陈维元，李英慧，张孟臣，景玉良，常汝镇，邱丽娟，. 绥农14及其系谱亲本的遗传多样性及重组分析[J]. 遗传, 2006, 28(11): 1421-1427.
[6]	刘乐承，向珣，曹家树. 白菜雄性不育相关基因BcMF4基因功能的RNAi验证[J]. 遗传, 2006, 28(11): 1428-1434.
[7]	唐丁，郭龙彪，曾大力，张光恒，程祝宽，钱前. 两例水稻极端异常分离现象的遗传分析[J]. 遗传, 2006, 28(10): 1259-1264.
[8]	张战凤，张鲁刚，王绮，惠麦侠，张明科. 利用RNA指纹技术分析大白菜胞质雄性不育相关基因的差异表达[J]. 遗传, 2006, 28(10): 1280-1286.
[9]	刘畅，杨足君，李光蓉，冯娟，邓科君，黄健，任正隆. 叶绿体基因infA-rpl36区域在小麦族物种中的序列变异分析[J]. 遗传, 2006, 28(10): 1265-1272.
[10]	曾兵，张新全，范彦，兰英，马啸，彭燕，刘伟. 鸭茅种质资源遗传多样性的ISSR研究[J]. 遗传, 2006, 28(9): 1093-1100.
[11]	姜金仲，李云，程金新. 植物同源四倍体生殖特性及DNA遗传结构的变异[J]. 遗传, 2006, 28(9): 1185-1190.
[12]	桂敏，曾亚文，杜娟，普晓英，申时全，杨树明，张浩. 粳稻孕穗期耐冷性NILs的形态评价和分子验证[J]. 遗传, 2006, 28(8): 972-976.
[13]	吴平治，李东屏. 拟南芥中MATE基因家族的研究进展 [J]. 遗传, 2006, 28(7): 906-910.
[14]	梅德圣，王汉中，李云昌，胡琼，李英德，徐育松. 甘蓝型油菜矮秆突变材料99CDAM的发现及遗传分析 [J]. 遗传, 2006, 28(7): 851-857.
[15]	刘传光，张桂权. 水稻单核苷酸多态性及其应用现状[J]. 遗传, 2006, 28(6): 737-744.