小菜蛾表皮蛋白基因克隆及表达分析.pdf

南方农业学报 Journal of Southern Agriculture2017,-48(12):2176-2181
ISSN 2095-1191: CODEN NXAAR
hilp://www.nlnyxb.com
1)Ol:10.3969jisn.2095-1191.2017.12.10
小菜蛾表皮蛋白基因克隆及表达分析
申建梅,陈炳輸,郭晓洁,胡黎明
、仲恺农业工程学院农业与生物学院,广州510225)
摘要:【日的】克隆小菜蛾表皮蛋白基因( Plutella xylostella cuticular protein, PXYICP),分析其序列特征和发育表
达模式,为深入研究PCP在小菜峨表皮形成中的生理功能提供科学参考。【方法)以小菜蛾为试验材料,采用反转
录聚合酶链式反应( R-PCR)和快速扩增cDNA末端(RACE)技术克降 Pxy/CP,以生物信息学方法对其序列特征进行
分析,并采用实时荧光定量PCR研究Pn/ CP MRNA在小菜蛾不同发育时期中的相对表达量。【结果】克隆获得的
Pxy/CP基因开放阅读框长531bp,编码177个织基酸,相对分子质量约18.90kD,等电点为5.32。氨基酸序列分析结果
表明,小菜蛾表皮蛋白第1~16位氮基酸是参与跨膜蛋白转移的信号肽,且该序列具有昆虫表皮蛋白CPR家族中RR-1
型保守基序的典型特征。不同发育时期的实时荧光定量PCR分析结果表明, PLCP在小菜峨各发育时期的表达量不
同,其中 Pxv/CP在2、3和4龄幼虫、蛹和成虫中的表达量分别是1龄幼虫的2.43、0.51、0.63、3.19和12.32倍,以在成虫的
表达量最高。【结论)成功克隆获得Px/CP基因,根据其发育表达模式推测 PXYICP蛋白参与小菜蛾成虫表皮的使化和
黑化等生理过程。
关键词:小菜蛾;表皮蛋白;基因克隆;实时荧光定量PCR
中图分类号
文献标志码:A
文章编号:2095-1191(2017)12-2176-06
Cloning and expression of Plutella xylostella( Linnaeus
cuticular protein gene
SHEN Jian-mei, CHEN Bing-han, GUO Xiao-jie, HU Li-ming
(College of Agriculture and Biology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China)
Abstract: [ Objective] Plutella ylostella( Linnaeus )cuticular protein gene(P/ P)was cloned, its sequence cha-
racters and developmental expression pattern were anal yzed to provides reference for the physiological function of Pxy/CP
in the formation of cuticular in P xylostella. [Method]P xylostella was used as materials, PICP was cloned by reverse
transcription polymerase chain reaction(RT-PCR )and rapid amplification CDNA ends(RACE). Py/CP sequence charac
ters were analyzed by bioinformatics method. The relative expression levels of Pxy/CP MRNA were investigated by real-
time fluorescent quantitative RTPCR method at different develomental stages. Result The cloned Py/CP contained 53 1
bp open reading frame in length, encoded 177 amino acids. Its relative molecular mass was 18. 90 KDA, and isoelectric
point was 5.32. Amino acid sequence analysis showed that the I to the 16"amino acids of cuticular protein was a signal
peptide that participated in transmembrane protein transfer. This sequence presented typical characteristics of RR-I conser-
vative base sequence in the CPR family of insect cuticular protein. According to real-time fluorescent quantitative PCR
analysis at various development stages, expression of Pxy /CP differed at each developmental stage. In 2 instar larva, 3
instar larva, 4 instar larva, pupal and adult, expression levels of Pxy/CP were 2.43, 0.51, 0.03, 3.19 and 12.32 times as
that in 1*instar larva. And the expression level reached the peak in adult. [Conclusion] Gene Pxr/CP is cloned successfull,
According to its development expression pattern, protein Pylcp is involved in the cuticle sclerotization and melanogen-
sis for P xylostella adu
Key words: Plutella xylostella(Linnaeus); cuticular protein; gene cloning; real-time quantitative PCR
0引言
us)为鱗翅目菜蛾科昆虫,主要危害十字花科蔬菜,
【研究意义】小菜蛾[ Plutella xylostella( Linac-致使十字花科作物的品质和产量大幅降低,甚至绝
收稿日期:2017-10-20
基金项目:国家自然科学基金青年科学基金项目(31401805);广东省高等学校优秀青年教师培养项目(KA1548850
作者简介:*为通讯作者,胡黎明(1978-),博上,副教授,主要从事昆虫分子生物学研究工作,E-mail:huining98(C126.com。申建
梅(1979-),博土,副教授,主要从事昆虫分子生物学研究工作,E-mail:shenjianmeil2163.com
万方数据