CN117925700A - GhTPS6基因在调控棉花黄萎病抗性中的应用 - Google Patents
GhTPS6基因在调控棉花黄萎病抗性中的应用 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域，特别是涉及GhTPS6基因在调控棉花黄萎病抗性中的应用。本发明通过VIGS实验验证了GhTPS6基因在抵御黄萎病方面的功能，在棉花中沉默GhTPS6基因会导致植株易感性提高、真菌含量增加和维管束褐化更加严重，表明GhTPS6基因在棉花对抗黄萎病中具有正调控作用。为今后棉花育种中提高棉花黄萎病抗性的有效基因资源的开发提供了基因资源。
Description
技术领域
本发明涉及基因工程技术领域，特别是涉及GhTPS6基因在调控棉花黄萎病抗性中的应用。
背景技术
植物可以产生多样化的次生代谢化合物，这些化合物作为防御产物，使植物能够适应环境，是植物在进化过程中与生物和非生物因素相互作用的结果。萜类化合物在功能和结构上是最多样化的次生代谢产物群体，目前已经鉴定出成千上万种萜类化合物。萜类化合物在植物与环境之间的交流中起着关键作用，并作为植物抗毒素防御病原体和食草动物，吸引传粉者和种子传播者，或通过传递诱惑或威慑信号诱导邻近植物的防御反应。
TPS基因广泛分布于植物中，它们的功能已经广泛研究，尤其是在抗病性方面。OsTPS19在水稻中被鉴定为(S)-柠檬烯合成酶，有效抑制了稻瘟病（Magnaporthe oryzae）孢子的萌发。过表达和RNAi实验表明，OsTPS19在对抗稻瘟病菌方面发挥了重要作用【Chen,X., Chen, H., Yuan, J.S., Köllner, T.G., Chen, Y., Guo, Y., Zhuang, X., Chen,X., Zhang, Y.J., Fu, J., Nebenführ, A., Guo, Z., Chen, F., 2018. The riceterpene synthase gene OsTPS19 functions as an (S)-limonene synthase inplanta, and its overexpression leads to enhanced resistance to the blastfungus Magnaporthe oryzae. Plant Biotechnol. J. 16, 1778-1787.】。在蒺藜苜蓿中，MtTPS10编码了一种倍半萜合成酶，突变体Mttps10植株比野生型植株更容易受到病原菌Aphanomyces euteiches感染【Yadav, H., Dreher, D., Athmer, B., Porzel, A.,Gavrin, A., Baldermann, S., Tissier, A., Hause, B., 2019. Medicago TERPENESYNTHASE 10 is involved in defense against an oomycete root pathogen. PlantPhysiol. 180, 1598-1613.】。
棉花是世界领先的可再生纺织纤维资源，也是食用油的重要来源。棉花的特征在于具有小而深色的色素腺体，含有倍半萜棉酚及其衍生物作为植物防御物质，提供了对害虫和病害的基本和诱导性保护。在棉花中已经鉴定出GhTPS1、GhTPS2和GhTPS3，机械损伤、真菌诱导剂和茉莉酸甲酯（MeJA）诱导了倍半萜的产生，同时也诱导了相应合成酶基因的表达【Yang, C.Q., Wu, X.M., Ruan, J.X., Hu, W.L., Mao, Y.B., Chen, X.Y., Wang,L.J., 2013. Isolation and characterization of terpene synthases in cotton(Gossypium hirsutum). Phytochemistry 96, 46-56.】。过表达GhTPS12的转基因烟草植株产生了相对较多的(3S)-芳樟醇，比野生型植株表现出对草食性昆虫更强的抵抗力【Huang, X.Z., Xiao, Y.T., Köllner, T.G., Jing, W.X., Kou, J.F., Chen, J.Y.,Liu, D.F., Gu, S.H., Wu, J.X., Zhang, Y.J., Guo, Y.Y., 2018. The terpenesynthase gene family in Gossypium hirsutum harbors a linalool synthaseGhTPS12 implicated in direct defence responses against herbivores. Plant CellEnviron. 41, 261-274.】。病毒诱导基因沉默（VIGS）实验证实了三个GhTPS基因与腺体活性有关【Zhang, C.P., Zhang, J.L., Sun, Z.R., Liu, X.Y., Shu, L.Z., Wu, H.,Song, Y., He, D.H., 2022. Genome-wide identification and characterization ofterpene synthase genes in Gossypium hirsutum. Gene 828, 146462.】。然而，TPS基因在对抗棉花最严重的土壤传播和导致血管枯萎的植物病原真菌黄萎病方面的作用很少被报道。
发明内容
为了解决上述问题，本发明提供了GhTPS6基因在调控棉花黄萎病抗性中的应用。本发明发现GhTPS6基因可以调控棉花黄萎病抗性，在棉花中沉默GhTPS6会导致植株易感性提高、真菌含量增加和维管束褐化更加严重。
为了实现上述目的，本发明提供了如下技术方案：
本发明提供了GhTPS6基因在调控棉花黄萎病抗性中的应用；所述GhTPS6基因编码的氨基酸序列如SEQ ID NO.12所示。
优选的，所述调控包括降低GhTPS6基因的表达量降低棉花黄萎病抗性。
优选的，所述GhTPS6基因的核苷酸序列如SEQ ID NO.13所示。
本发明提供了一种降低棉花黄萎病抗性的重组载体，包括GhTPS6基因的部分序列和pTRV2载体；所述GhTPS6基因编码的氨基酸序列如SEQ ID NO.12所示。
优选的，所述GhTPS6基因的部分序列的核苷酸序列如SEQ ID NO.5所示。
本发明提供了一种降低棉花黄萎病抗性的工程菌，包括原始菌和上述技术方案所述的重组载体。
优选的，所述原始菌包括农杆菌（Agrobacterium）。
优选的，所述农杆菌包括根癌农杆菌（Agrobacterium tumefaciens）。
本发明提供了上述技术方案所述的重组载体或上述技术方案所述的工程菌在降低棉花黄萎病抗性中的应用。
有益效果：
本发明提供了GhTPS6基因在调控棉花黄萎病抗性中的应用；所述GhTPS6基因编码的氨基酸序列如SEQ ID NO.12所示。本发明通过VIGS实验验证了GhTPS6基因在抵御黄萎病方面的功能，在棉花中沉默GhTPS6基因会导致植株易感性提高、真菌含量增加和维管束褐化更加严重，表明GhTPS6基因在棉花对抗黄萎病中具有正调控作用。为今后棉花育种中提高棉花黄萎病抗性的有效基因资源的开发提供了基因资源。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍。
图1为在烟草叶片中GhTPS6-GFP融合蛋白的亚细胞定位结果；
图2为pTRV2-GhCLA1接种植株16天后的白化表型和qRT-PCR检测GhTPS基因的沉默效率；
图3为棉花中GhTPS基因的沉默后植株对黄萎病的抗性结果。
具体实施方式
本发明提供了GhTPS6基因在调控棉花黄萎病抗性中的应用；所述GhTPS6基因编码的氨基酸序列如SEQ ID NO.12所示，具体如下：MASQVSQILASPHTSIPCNMENRPKADFHPGIWGDIFLNYPNEDIDTATQLRHEELKEEVRRKLVAPMGDNSTQKLPLIDAVQRLGVYYHFEEEIEDALEAIYHDNNDVDNDLYTTSLRFRLLREHGFNVSCEVFDKFKDEEGNFMSSITTDVQGLLELYEASYMRVHGEDILDEAISFTTTHITLAAPTLEYPLSEQVAHALKQSIRRGLPRVEARRYISIYQDIESHNKALLEFAKIDFNLLQLLHRKELSEICRWWKDLDFTKKLPFARDRVVEGYFWIMGVYFEPQYSLGRKMMTKVIAMASIVDDTYDSYATYDELIPYTDAIERWDIKCMDELPEFMKISYKALLDVYEEMEQLMAKQGRQYRVKYARKAMIRLAQAYLLEAKWTHQNYKPTFEEFRDNALPTSGYGMLAITAFVGMGDVITPETFEWATNDPKIIKASTIICRFMDDIAEHKFKHRREDDCSAIECYMEQYGVTAQEAYDEFNKHIESSWKDINEEFLKPTEMPVPVLNRSLNLARVMDVLYREGDGYTHVGKAAKGGISSLLIDPIPL；
在本发明中，所述GhTPS6基因的核苷酸序列优选如SEQ ID NO.13所示，具体如下：5’-ATGGCTTCACAAGTTTCTCAAATCCTTGCTTCACCCCATACTTCCATTCCCTGCAACATGGAAAATCGTCCCAAGGCTGATTTTCACCCTGGCATTTGGGGAGATATCTTCCTCAATTACCCTAATGAGGATATTGATACTGCAACTCAACTCCGACATGAAGAACTAAAAGAAGAAGTTAGGCGGAAGCTTGTGGCTCCCATGGGTGATAATTCAACCCAAAAACTGCCCCTTATTGATGCAGTCCAAAGGTTGGGTGTGTATTACCATTTTGAGGAAGAGATTGAAGATGCATTAGAAGCTATATACCATGACAACAATGATGTCGACAATGATCTCTATACCACCTCTCTTCGATTTCGATTACTTAGAGAGCATGGCTTTAATGTTTCATGTGAGGTATTCGATAAGTTCAAAGATGAGGAAGGAAATTTCATGTCATCCATAACCACTGATGTGCAAGGACTGTTGGAGCTTTATGAAGCTTCGTATATGCGGGTGCATGGGGAAGATATATTGGATGAGGCAATTTCTTTCACCACCACTCACATAACTCTTGCAGCACCGACTTTAGAGTATCCATTGTCGGAACAAGTTGCACATGCCTTAAAACAGTCCATCCGAAGAGGCTTGCCTAGGGTCGAGGCTAGGCGATACATTTCGATATACCAGGATATTGAATCCCATAATAAGGCATTGTTGGAATTTGCAAAGATAGATTTCAACTTGTTACAGCTTTTGCATAGAAAAGAGCTAAGTGAAATTTGTAGGTGGTGGAAGGATTTAGACTTTACAAAAAAGTTACCGTTTGCGAGAGATAGAGTGGTTGAAGGTTACTTTTGGATAATGGGAGTATACTTTGAGCCACAATACTCTCTTGGTAGAAAGATGATGACAAAAGTAATAGCCATGGCATCTATCGTAGATGATACATATGACTCATATGCAACATATGATGAACTCATTCCCTATACAGATGCAATCGAGAGGTGGGATATTAAATGTATGGATGAGCTCCCAGAATTCATGAAAATAAGCTATAAAGCACTATTAGATGTTTATGAGGAAATGGAGCAACTAATGGCAAAGCAAGGGAGACAATATCGTGTCAAATATGCAAGGAAGGCGATGATACGACTTGCTCAAGCTTACCTTTTGGAAGCCAAATGGACCCATCAAAACTACAAACCAACATTTGAGGAGTTTAGAGATAATGCATTGCCAACCTCGGGCTATGGCATGCTTGCTATTACAGCTTTTGTGGGCATGGGAGATGTTATAACTCCAGAGACATTTGAATGGGCAACCAACGACCCTAAGATCATCAAAGCTTCCACAATAATTTGCAGGTTCATGGATGACATCGCTGAACACAAGTTCAAGCATCGGAGAGAAGATGATTGCTCAGCCATCGAGTGCTACATGGAACAATATGGCGTAACGGCACAAGAGGCTTATGATGAATTCAACAAGCATATAGAGAGCTCCTGGAAGGACATTAACGAAGAGTTCCTGAAACCAACAGAAATGCCAGTACCTGTTCTAAATAGAAGTCTCAACCTTGCAAGAGTGATGGATGTGCTTTACAGAGAAGGAGATGGTTATACACATGTTGGCAAAGCTGCTAAAGGTGGGATCAGTTCATTGCTCATCGATCCAATTCCACTTTGA-3’。
在本发明中，所述调控优选包括降低GhTPS6基因的表达量降低棉花黄萎病抗性。本发明通过VIGS实验验证了GhTPS6基因在抵御黄萎病方面的功能，在棉花中沉默GhTPS6基因会导致植株易感性提高、真菌含量增加和维管束褐化更加严重，表明GhTPS6基因在棉花对抗黄萎病中具有正调控作用。
本发明提供了一种降低棉花黄萎病抗性的重组载体，包括GhTPS6基因的部分序列和pTRV2载体；所述GhTPS6基因编码的氨基酸序列如SEQ ID NO.12所示。
在本发明中，所述GhTPS6基因的部分序列的核苷酸序列优选如SEQ ID NO.5所示。
本发明提供了一种降低棉花黄萎病抗性的工程菌，包括原始菌和上述技术方案所述的重组载体。在本发明中，所述原始菌优选包括农杆菌；所述农杆菌优选包括根癌农杆菌，更优选为根癌农杆菌GV3101。
本发明提供了上述技术方案所述的重组载体或上述技术方案所述的工程菌在降低棉花黄萎病抗性中的应用。本发明提供的重组载体或工程菌可以降低GhTPS6基因的表达量，从而降低棉花黄萎病抗性。
为了进一步说明本发明，下面结合附图和实施例对本发明提供的GhTPS6基因在调控棉花黄萎病抗性中的应用进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
实施例1
利用ClonExpress® Ultra One Step Cloning Kit将去掉终止密码子的GhTPS6基因的CDS与pCAMBIA2300-GFP载体中的GFP进行同源重组，得到重组质粒35S::GhTPS6-GFP，具体方法如下：
1. 目的片段的获得：以TM-1棉花的cDNA为模板，利用TAKARA的高保真酶PrimeSTAR® GXL Premix扩增序列，PCR反应体系如下：PrimeSTAR GXL Premix 25 µL，引物2300-GhTPS6-F 1.5 µL，引物2300-GhTPS6-R 1.5 µL，模板2 µL，ddH2O 20 µL；所述引物2300-GhTPS6-F的核苷酸序列如SEQ ID NO.1所示，具体如下：5’-ATACTAGTGGATCCGGTACCATGGCTTCACAAGTTTCTCAAATCC-3’；所述引物2300-GhTPS6-R的核苷酸序列如SEQ ID NO.2所示，具体如下：5’-CCCTTGCTCACCATGGTACCAAGTGGAATTGGATCGATGAG-3’；
PCR反应条件如下：98 ℃预变性5 min；98 ℃变性10 s、58 ℃退火15 s，68 ℃延伸1 min/kb，共35个循环；68 ℃终延伸10 min。
2. 表达载体线性化：将所用表达载体在37 ℃下孵育3 h双酶切以获得线性化载体，反应体系为：pCAMBIA2300-GFP 1 μg，BamH Ⅰ 1 µL，Sac Ⅰ 1 µL，CutSmart 5 µL，ddH2O补足至50 µL。
3. 将上述PCR产物和线性化表达载体通过北京全式金EasyPure® Quick GelExtraction Kit回收纯化。
4. 利用南京诺唯赞的ClonExpress® Ultra One Step Cloning Kit将线性化载体和目的片段进行同源重组，构建载体，冰上配制以下体系：线性化载体1 µL，目的片段4 µL，2×ClonExpress Mix 5 µL。50 ℃反应10 min，立即置于冰上冷却。然后将重组产物转化进大肠杆菌感受态中。测序比对成功后使用北京全式金EasyPure® Plasmid MiniPrep Kit进行大肠杆菌质粒的提取。
将重组质粒35S::GhTPS6-GFP和空的pCAMBIA2300-GFP载体转化到根癌农杆菌菌株GV3101，通过注射5周龄烟草叶片至80%的叶片湿润，进行瞬时表达。然后通过激光扫描共聚焦显微镜检测GFP荧光信号。结果见图1，比例尺=20 μm。
由图1可知，在细胞核和细胞质中均观察到GhTPS6-GFP融合蛋白的GFP荧光。
实施例2
植物材料与病毒诱导的基因沉默（VIGS）
1. 目的片段的获得：以TM-1棉花的cDNA为模板，利用TAKARA的高保真酶PrimeSTAR® GXL Premix扩增序列，PCR反应体系如下：PrimeSTAR GXL Premix 25 µL，引物VIGS-GhTPS6-F 1.5 µL，引物VIGS-GhTPS6-R 1.5 µL，模板2 µL，ddH2O 20 µL；所述引物VIGS-GhTPS6-F的核苷酸序列如SEQ ID NO.3所示，具体如下：5’-gtgagtaaggttaccgaattcTGATTTTCACCCTGGCATTTG-3’；所述引物VIGS-GhTPS6-R的核苷酸序列如SEQ ID NO.4所示，具体如下：5’-cgtgagctcggtaccggatccTGCTCTCTAAGTAATCGAAATCGAA-3’；
PCR反应条件如下：98 ℃预变性5 min；98 ℃变性10 s、58 ℃退火15 s，68 ℃延伸1 min/kb，共35个循环；68 ℃终延伸10 min。
2. 表达载体线性化：将所用表达载体在37 ℃下孵育3 h双酶切以获得线性化载体，反应体系为：pTRV2 1 μg，BamH Ⅰ 1 µL，EcoR Ⅰ 1 µL，CutSmart 5 µL，ddH2O补足至50µL。
3. 将上述PCR产物和线性化表达载体通过北京全式金EasyPure® Quick GelExtraction Kit回收纯化；所述PCR产物的核苷酸序列如SEQ ID NO.5所示，具体如下：5’-TGATTTTCACCCTGGCATTTGGGGAGATATCTTCCTCAATTACCCTAATGAGGATATTGATACTGCAACTCAACTCCGACATGAAGAACTAAAAGAAGAAGTTAGGCGGAAGCTTGTGGCTCCCATGGGTGATAATTCAACCCAAAAACTGCCCCTTATTGATGCAGTCCAAAGGTTGGGTGTGTATTACCATTTTGAGGAAGAGATTGAAGATGCATTAGAAGCTATATACCATGACAACAATGATGTCGACAATGATCTCTATACCACCTCTCTTCGATTTCGATTACTTAGAGAGCA-3’。
4. 利用南京诺唯赞的ClonExpress® Ultra One Step Cloning Kit将线性化载体和目的片段进行同源重组，构建载体，冰上配制以下体系：线性化载体1 µL，目的片段4 µL，2×ClonExpress Mix 5 µL。
50 ℃反应10 min，立即置于冰上冷却。然后将重组产物转化进大肠杆菌感受态中。测序比对成功后使用北京全式金EasyPure® Plasmid MiniPrep Kit进行大肠杆菌质粒的提取，得到TRV::GhTPS6重组载体。
5. 农杆菌转化
1）向刚刚融化的农杆菌感受态GV3101中加入1 µL的质粒，轻轻混匀。
2）冰上静置10 min，随后将离心管置于液氮中速冻5 min。
3）快速将离心管放入37 ℃水浴5 min，不要晃动水面。
4）再置于冰上静置5 min。
5）加入800 µL不添加抗生素的LB液体培养基，置于28 ℃摇床摇2~3 h。
6）5,000 r/min离心2 min，在超净工作台中弃700 µL上清液，留下100 µL菌液吹打均匀涂布于含有K+抗生素的LB固体培养基上，吹干后倒置于28 ℃培养箱中培养2~3 d。
6. 棉花幼苗的VIGS处理
在26 ℃的温室中，16小时光照/8小时黑暗循环，相对湿度约为70%，培养对黄萎病具有抗性的陆地棉品种9053的种子，待棉花幼苗生长至子叶平展期时，选取生长较为一致的幼苗，利用注射法对幼苗子叶进行VIGS处理。
1）在对棉花幼苗进行VIGS处理前2天，分别取保存于-80 ℃的辅助载体pTRV1，空载体pTRV2（阴性对照），阳性对照pTRV2-GhCLA1（参见【王心宇, 吕坤, 蔡彩平, 徐君, 郭旺珍, 2014. TRV病毒介导的基因沉默体系在棉花中的建立及应用. 作物学报 40, 8.】）和目的基因TRV::GhTPS6重组载体菌液50 μL，置于2 mL无菌离心管中，并向离心管中加入500 μL灭菌LB（含卡那霉素（K+）50 μg/mL和利福平（Rif+）50 μg/mL）培养液，于28 ℃摇床200 r/min活化培养。
2）将活化好的菌株接种于含50 mL LB培养液（含K+& Rif+）的灭菌三角瓶中，并置于28 ℃，200 r/min 摇床培养16 h。
3）摇床培养农杆菌菌液至OD600值为0.8左右时，将菌液收集于灭菌处理过的50 mL离心管中，8000 r/min 离心10 min。去除离心管中的上清液后，使用VIGS重悬液重新调整菌液OD600值至0.8~1.0，并将阳性对照pTRV2-GhCLA1、目的基因TRV::GhTPS6重组载体和pTRV2空载体的重悬菌液分别与辅助载体pTRV1重悬菌液按1:1等体积混匀，室温下放置3h。
VIGS 菌液重悬液配方为：氯化镁MgCl：2.03 g/L、吗啉乙磺酸MES：2.135 g/L和乙酰丁香酮AS：0.03 g/L；
5）在进行VIGS处理时，首先用1 mL枪头在棉花幼苗子叶背面，轻点3~4个小孔。然后用1 mL注射器将处理好的菌液延着小孔注射到棉花子叶，直至整个子叶呈现出水渍状。待幼苗VIGS处理完毕后，统一浇水，并用黑色塑料膜将处理好的棉花幼苗覆盖，避光培养24h。
6）VIGS处理幼苗子叶第二天，将黑色塑料膜揭开，保持培养条件为25 ℃，16 h 光照/8 h黑暗。
黄萎病菌培养及接种
将三亚中国农业科学院国家南繁研究院本实验室保藏的黄萎病菌株V592（公开于【Sun, M., Zhang, Z., Ren, Z., Wang, X., Sun, W., Feng, H., Zhao, J., Zhang,F., Li, W., Ma, X., Yang, D., 2021. The GhSWEET42 Glucose transporterparticipates in Verticillium dahliae infection in cotton. Front Plant Sci 12,690754.】）在马铃薯葡萄糖琼脂（PDA）培养基上25 ℃黑暗培养4~5天，然后转移至Czapek培养基中，25 ℃下摇床中120 rpm摇3天。当pTRV2-GhCLA1植株出现叶片白化表型时，利用去离子水将菌液孢子浓度调至107/mL，采用灌根法接种黄萎病。对照组给予相同体积的Czapek培养基处理。每个处理至少使用25株植株，每个处理重复三次。
黄萎病接种后的疾病评估
接种后16天，收集棉花的根用于评估幼苗真菌生物量和GhTPS6基因的相对表达量。采用真菌特异性引物ITS1-F和大丽轮枝菌特异性反向引物STVe1-R进行实时荧光定量PCR（qRT-PCR），qRT-PCR-GhTPS6-F和qRT-PCR-GhTPS6-R检测GhTPS6基因的相对表达量，以GhHis3为内参，引物序列如下：
ITS1-F：5’-AAAGTTTTAATGGTTCGCTAAGA-3’，SEQ ID NO.6；
STVe1-R：5’-CTTGGTCATTTAGAGGAAGTAA-3’，SEQ ID NO.7；
GhHis3-F：5’-GACACCAACCTTTGCGCGAT-3’，SEQ ID NO.8；
GhHis3-R：5’-AGCGACTGATCCACACTTCTG-3’，SEQ ID NO.9；
qRT-PCR-GhTPS6-F：5’-GTGAAATTTGTAGGTGGTGGAAGG-3’，SEQ ID NO.10；
qRT-PCR-GhTPS6-R：5’-ACCACTCTATCTCTCGCAAACG-3’，SEQ ID NO.11；
所述qRT-PCR的反应体系为：10 µL 2×ChamQ Universal SYBR qPCR MasterMix，引物F/R各0.5 µL，模板cDNA 1 µL，加ddH2O至20 µL ；
所述qRT-PCR的反应程序为：预变性95 ℃，30 s；循环反应95 ℃，10 s，60 ℃，30s，40个循环；融解曲线使用仪器默认程序；结果见图2、图3中的A和图3中的B，其中，图2中的A为pTRV2-GhCLA1接种植株16天后的白化表型；图2中的B为qRT-PCR检测GhTPS基因的沉默效率结果；图3中的A为接种16天后的GhTPS基因沉默植株和阴性对照植株（TRV::00）的病害症状；图3中的B为qRT-PCR测定TRV::00和TRV::GhTPS植株中真菌生物量结果；**为P <0.01。
收集每株植物同一位置子叶节上的新鲜茎，用于大丽轮枝菌恢复试验。茎部用75%酒精浸泡1分钟，再用30%双氧水浸泡30分钟，用无菌水冲洗4~5次。将切成1 cm的茎段置于PDA培养基上，25 ℃培养3天，观察大丽轮枝菌的生长情况，观察方法参见【Sun, M.,Zhang, Z., Ren, Z., Wang, X., Sun, W., Feng, H., Zhao, J., Zhang, F., Li, W.,Ma, X., Yang, D., 2021. The GhSWEET42 glucose transporter participates inVerticillium dahliae infection in cotton. Front Plant Sci. 12, 690754.】。疾病指数（Disease index, DI）评分范围从0（无）到4（严重）用于评估疾病严重程度，DI的计算方法参见【Gong, Q., Yang, Z., Chen, E., Sun, G., He, S., Butt, H.I., Zhang,C., Zhang, X., Yang, Z., Du, X., Li, F., 2018. A phi-class glutathione S-transferase gene for Verticillium wilt resistance in Gossypium arboreumidentified in a genome-wide association study. Plant Cell Physiol. 59, 275-289.】。结果见图3中的C~E，其中，图3中的C为TRV::00和TRV::GhTPS植株的病情指数，图3中的D为真菌恢复实验结果，图3中的E为接种黄萎病的TRV::00和TRV::GhTPS植株的维管组织。
由图2可知，当pTRV2-GhCLA1植株出现白化叶片表型时，qRT-PCR分析显示与对照植株相比，接种TRV::GhTPS6重组载体的植株中每个目标基因（GhTPS6）的表达水平显著降低，证明了基因沉默的有效性。
由图3可知，棉花幼苗接种黄萎病V592菌株16天后GhTPS6沉默植株与阴性对照植株相比表现出更多的萎蔫和萎黄（图3中的A）。真菌生物量的qRT-PCR分析显示GhTPS6沉默植株中真菌含量较高（图3中的B）。GhTPS6沉默植株的病害指数显著高于对照植株（图3中的C）。在接种棉花的茎段上进行的真菌恢复实验显示，TRV::GhTPS6植株的菌丝生长明显，而对照植株几乎没有菌丝生长（图3中的D）。与此发现一致，TRV::GhTPS6植株的维管束变褐现象比TRV::00植株更严重（图3中的E）。综上所述，沉默了GhTPS6基因的棉花植株更易感病，GhTPS6基因在调控棉花对黄萎病的抗性中起关键作用。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。
Claims (9)
1.GhTPS6基因在调控棉花黄萎病抗性中的应用；所述GhTPS6基因编码的氨基酸序列如SEQ ID NO.12所示。
2.根据权利要求1所述的应用，其特征在于，所述调控包括降低GhTPS6基因的表达量降低棉花黄萎病抗性。
3.根据权利要求1或2所述的应用，其特征在于，所述GhTPS6基因的核苷酸序列如SEQID NO.13所示。
4.一种降低棉花黄萎病抗性的重组载体，其特征在于，包括GhTPS6基因的部分序列和pTRV2载体；所述GhTPS6基因编码的氨基酸序列如SEQ ID NO.12所示。
5.根据权利要求4所述的重组载体，其特征在于，所述GhTPS6基因的部分序列的核苷酸序列如SEQ ID NO.5所示。
6.一种降低棉花黄萎病抗性的工程菌，其特征在于，包括原始菌和权利要求4或5所述的重组载体。
7.根据权利要求6所述的工程菌，其特征在于，所述原始菌包括农杆菌（Agrobacterium）。
8.根据权利要求7所述的工程菌，其特征在于，所述农杆菌包括根癌农杆菌（Agrobacterium tumefaciens）。
9.权利要求4或5所述的重组载体或权利要求6~8任一项所述的工程菌在降低棉花黄萎病抗性中的应用。
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