中国农业科学 ›› 2019, Vol. 52 ›› Issue (3): 466-477.doi: 10.3864/j.issn.0578-1752.2019.03.007
唐斌1,2,沈祺达2,曾伯平1,肖仲久1,邱玲玉2,潘碧莹2,李昆1,张道伟1()
收稿日期:
2018-09-11
接受日期:
2018-10-23
出版日期:
2019-02-01
发布日期:
2019-02-14
作者简介:
唐斌,E-mail: 基金资助:
TANG Bin1,2,SHEN QiDa2,ZENG BoPing1,XIAO ZhongJiu1,QIU LingYu2,PAN BiYing2,LI Kun1,ZHANG DaoWei1()
Received:
2018-09-11
Accepted:
2018-10-23
Online:
2019-02-01
Published:
2019-02-14
摘要:
背景 昆虫海藻糖合成酶基因是昆虫海藻糖合成的主要基因,大多数昆虫中只拥有一个海藻糖-6-磷酸合成酶(trehalose-6-phosphate synthase,TPS)基因,部分昆虫存在一个海藻糖-6-磷酸酯酶(trehalose-6-phosphate phosphatase,TPP)基因。前期研究发现褐飞虱(Nilaparvata lugens)中拥有两个TPS,对其功能研究发现TPS不仅能够调控海藻糖代谢,还可介导海藻糖酶调控几丁质合成与降解途径,控制昆虫的蜕皮过程。目的 通过对褐飞虱转录组测序分析获得了一个新的TPS,检测该基因在褐飞虱不同发育阶段的表达情况,探究该基因的功能与前期发现的两个TPS的区别。方法 对获得的新TPS基因序列采用克隆技术获得全长cDNA序列,经验证正确后,对其蛋白的一级、二级、三级结构及与其他昆虫的TPS进行比对分析,最后采用实时荧光定量PCR(qRT-PCR)技术测定3个不同TPS在褐飞虱不同发育阶段的表达,并采用RNA干扰(RNAi)技术抑制TPS3的表达。结果 在前期研究的基础上,克隆出一个新的TPS,并命名为TPS3。TPS3开放阅读框长度为2 352 bp,编码783个氨基酸,预测蛋白分子量为88.9 kD,等电点为5.47,具有亲水性结构。生物信息学分析表明,褐飞虱3个TPS蛋白具有较高的同源性,都具有TPS和TPP两个保守结构域及其他特征序列,并且α-螺旋、β-折叠和无规则卷曲所占的比例较为接近。褐飞虱不同发育阶段3条TPS的相对表达量不同,TPS1的相对表达量从4龄0 h开始逐渐上升,至成虫阶段达到最高,TPS2的相对表达量从4龄末期开始明显上升且在整个5龄阶段都有较高的表达,TPS3的相对表达量在5龄末期和成虫初期较高。单独干扰褐飞虱TPS3 48 h后被干扰基因的相对表达量下降,dsTPS3能有效抑制TPS3的表达。结论 在褐飞虱中发现一个新的TPS(TPS3),其与褐飞虱中已经报道的TPS1和TPS2具有较高的同源性。不同发育阶段表达结果表明,3个TPS在发育过程中行使的功能不同。RNAi能够有效抑制TPS3的表达并导致褐飞虱蜕皮障碍和翅发育畸形。
唐斌,沈祺达,曾伯平,肖仲久,邱玲玉,潘碧莹,李昆,张道伟. 褐飞虱一个新的海藻糖合成酶基因的特性、 发育表达及RNAi效果分析[J]. 中国农业科学, 2019, 52(3): 466-477.
TANG Bin,SHEN QiDa,ZENG BoPing,XIAO ZhongJiu,QIU LingYu,PAN BiYing,LI Kun,ZHANG DaoWei. Characteristics, Developmental Expression and RNAi Effect Analysis of a Novel Trehalose-6-Phosphate Synthase Gene in Nilaparvata lugens[J]. Scientia Agricultura Sinica, 2019, 52(3): 466-477.
表1
荧光实时定量PCR引物和TPS3及GFP的dsRNA引物序列"
基因名称 Primer name | 正向引物 Forward primer (5′-3′) | 反向引物 Reverse primer (5′-3′) |
---|---|---|
QNl18S QNlTPS1 QNlTPS2 QNlTPS3 | CGCTACTACCGATTGAA AAGACTGAGGCGAATGGT AGAGTGGACCGCAACAACA GTGATGCGTCGGTGGCTAT | GGAAACCTTGTTACGACTT AAGGTGGAAATGGAATGTG TCAACGCCGAGAATGACTT CCGTTCATCATTGGGCATAGT |
dsNlTPS3 | GAGTCTGACCTGATAGCCTTTA | ATCGGAGTCCATTTAGTTGT |
dsNlTPS3-T7 | GGATCCTAATACGACTCACTATAGGGAGTCTGACCTGATAGCCTTTA | GGATCCTAATACGACTCACTATAGGATCGGAGTCCATTTAGTTGT |
dsNlGFP | AAGGGCGAGGAGCTGTTCACCG | CAGCAGGACCATGTGATCGCGC |
dsNlGFP-T7 | GGATCCTAATACGACTCACTATAGGAAGGGCGAGGAGCTGTTCACCG | GGATCCTAATACGACTCACTATAGGCAGCAGGACCATGTGATCGCGC |
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