玉米萜类合成酶基因tps2的功能及其启动子功能区段鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2020-0563

摘要/Abstract

摘要：

虫害是造成玉米减产和品质下降的主要原因,玉米萜类合成酶基因tps2（terpene synthase 2）受虫害诱导表达,催化产生多种萜类化合物。qPCR检测不同激素和黏虫处理条件下tps2的表达;过表达植株（TPS2OE）进行玉米螟行为选择实验;原生质体瞬时表达检测tps2启动子的活性。结果显示植物激素（JA、SA、ET）和黏虫诱导tps2高表达;TPS2OE中tps2的表达量及目的萜类含量显著高于对照B73;玉米螟行为选择实验结果表明,尽管实验组和对照组叶片上的玉米螟卵的个数无显著差异,但TPS2OE叶片上的幼虫数较少,危害面积也更小;启动子截短分析发现-183至-129为tps2启动子必需的功能区段,并且-227至-183区段可能存在负调控元件。综上,tps2受植物激素和虫害诱导表达,TPS2OE植株对玉米螟幼虫有一定的抑制作用,tps2启动子必需的功能区段为-183至-129。

关键词: 玉米, tps2, 萜类化合物, 植物激素, 亚洲玉米螟, 启动子

Abstract:

Insect pest is the main reason causing the yield and quality of corn reduced. The tps2（terpene synthase 2）gene is inducible by herbivore and TPS2 catalyzes the production of many terpenoids. To analyze the function of tps2 and its expression regulation mechanism,qPCR was used to detect the expression of tps2 under different hormones and armyworm treatments. The transgenic maize TPS2OE was obtained by Agrobacterium-mediated transformation using a tps2 overexpression vector and it was used to study the selective behavior of Asian corn borer. The functional segment of tps2 promoter was tested by transient expression system of maize protoplast. The results showed that plant hormones（JA,SA,and ET）and armyworm induced high expression of tps2;and the expression of tps2 in TPS2OE and the target terpenoids were significantly higher than that in control B73. The number of corn borer eggs on the leaves of TPS2OE and B73 was insignificant,while the larvae on TPS2OE were fewer,and the damage areas were also smaller. The promoter truncation analysis found that the required functional element of the tps2 promoter was between -183 to -129,and there were repressor binding sites between -227 to -183. In summary,tps2 expression is induced by plant hormones and armyworm,and TPS2OE plants have a certain inhibitory effect on the larvae of Asian corn borer. The required functional element of tps2 promoter is between -183 to -129.

Key words: maize, tps2, terpenoids, plant hormones, Asian corn borer, promoter

王丹, 李圣彦, 刘进平, 郎志宏. 玉米萜类合成酶基因tps2的功能及其启动子功能区段鉴定[J]. 生物技术通报, 2020, 36(12): 1-11.

WANG Dan, LI Sheng-yan, LIU Jin-ping, LANG Zhi-hong. Study on the Function of Terpene Synthase Gene tps2 and Its Promoter Functional Segment in Zea mays[J]. Biotechnology Bulletin, 2020, 36(12): 1-11.

图/表 11

图1 玉米螟行为选择实验设计

表1 用于扩增不同长度tps2启动子的引物序列

引物名称	引物序列（5'-3'）
tps2Pwf-R	GCGGCCGCTCTAGAACTAGTGGATCCCGTCT- ACTACTCGAACTAACTGAAC
tps2Pwf968-F	TCGAGGTCGACGGTATCGATAAGCTTGTAC- ACACATCATCAATAAG
tps2Pwf737-F	TCGAGGTCGACGGTATCGATAAGCTTAACTG- TGGTCGTCGCAAT
tps2Pwf485-F	TCGAGGTCGACGGTATCGATAAGCTTCGTTTT- TTTATCCATTGC
tps2Pwf327-F	TCGAGGTCGACGGTATCGATAAGCTTCTGTA- TGCCCTGACCTGA
tps2Pwf227-F	TCGAGGTCGACGGTATCGATAAGCTTCTGGC- ATCGGAAGAAGCT
tps2Pwf183-F	TCGAGGTCGACGGTATCGATAAGCTTTCGCC- CTATAAATTGGAG
tps2Pwf129-F	TCGAGGTCGACGGTATCGATAAGCTTTCACA- ACACAAGCAGGCA
tps2Pwf61-F	TCGAGGTCGACGGTATCGATAAGCTTGGCTTG- CTTTGGTTTCTG

图2 玉米萜类合成酶进化树分析 Tps：terpene synthase;stc1：sesquiterpene cyclase 1;an1：anther ear1;cpps2：copalyl diphosphate synthase 2

图3 玉米TPS2和其他物种的萜类合成酶系统发育树 TPS：terpene synthase;TPC：terpenoid cyclases;TPSL：terpene synthase-like sequence;HLS：Alpha-humulene synthase;;GERD：（-）-germacrene D synthase;BLS：（S）-beta-bisabolene synthase;ATS：alpha-terpineol synthase;NLS：nerolidol synthase;MCS：（S）-beta-macrocarpene synthase;CBS：Beta-cubebene synthase;LLS：linalool synthase。Zm：玉米;Sob：双色高粱;LOC Os：水稻;Bra：二穗短柄草;Sei：狗尾草;At：拟南芥

图4 不同植物激素（JA、SA和ET）和黏虫（AW）诱导tps2基因表达分析

图5 在转录水平上B73和TPS2OE植株中tps2基因的表达情况误差线代表标准误差,“**”表示P <0.01

图6 cyp92c5和cyp92c6基因在B73和TPS2OE中的表达情况

图7 TPS2OE和B73玉米中TMTT的GC-MS分析 A：TPS2OE挥发物的气相色谱图;B：B73挥发物的气相色谱图;C：TMTT的质谱图

图8 玉米螟对B73和TPS2OE幼苗的行为选择 A：两组玉米幼苗的产卵个数;B：两组玉米幼苗上发现的幼虫数量;C：两组玉米幼苗受到的危害面积（误差棒代表标准误差,“*”表示P <0.05,有显着差异）

图9 截短tps2启动子系列载体示意图 A：PCR扩增tps2启动子片段;B：载体示意图

图10 LUC双荧光分析tps2启动子活性

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