CN115976033A - 瓜实蝇tssk1和tssk3基因及其应用 - Google Patents
瓜实蝇tssk1和tssk3基因及其应用 Download PDFInfo
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Abstract
本发明涉及分子生物学技术领域,具体涉及瓜实蝇TSSK1和TSSK3基因及其应用。本发明要解决的技术问题是为瓜实蝇的绿色可持续防治提供一种新选择。本发明的技术方案是瓜实蝇TSSK1基因,其核苷酸序列如SEQ ID No.1所示。本发明还提供了瓜实蝇TSSK3基因,其核苷酸序列如SEQ ID No.2所示。该基因可用于降低瓜实蝇雄虫生殖力,为解决瓜实蝇在化学防治过程中产生的各种环境问题及瓜实蝇绿色防控提供新思路,为利用SIT防治瓜实蝇提供潜在靶标。
Description
技术领域
本发明涉及分子生物学技术领域,具体涉及瓜实蝇ZcTSSK1和ZcTSSK3基因及其应用。
背景技术
瓜实蝇Zeugodacus cucurbitae(Coquillett),是一种广泛分布于温带、热带以及亚热带地区的经济害虫。危害近130多种寄主植物,特别是葫芦科作物,严重影响我国果蔬产业的健康发展。由于瓜实蝇食性杂,寄主范围比较广泛,有很强的繁殖和迁飞能力,传播速度较快,所以有效防治相对困难,导致爆发成灾,给农业生产造成较大威胁,给瓜果农造成重大的经济损失。目前,对瓜实蝇的主要防治方式仍然以化学防治为主,该防治手段效果明显,但易产生抗药性,对环境造成污染,而且瓜实蝇主要是在寄主植物内部产卵危害,使用化学药剂效果非常有限。
昆虫不育技术(sterile insect techniques,SIT)是指通过向田间释放经处理后无生殖力且与野生型雄性有竞争力的雄性昆虫,使之能与野生型雌虫交配,减少野生型雄虫的交配数量,从而达到减少子代数量的一种绿色的害虫防治方法。该种方法在很多地方的多种昆虫中都成功应用,并取得了不错的效果。越来越多的研究试图阐明控制昆虫精子发生的基因和分子过程,以确定可作为潜在SIT靶点的关键基因。确定精子发生所必需的关键基因的进一步研究也是有必要的,并为指导进一步完善男性不育技术提供了机会。
发明内容
本发明要解决的技术问题是为瓜实蝇的绿色防控提供一种新选择。
本发明的技术方案是瓜实蝇TSSK1基因,其核苷酸序列如SEQ ID No.1所示。
本发明还提供了瓜实蝇TSSK3基因,其核苷酸序列如SEQ ID No.2所示。
本发明还提供了所述瓜实蝇TSSK1或TSSK3基因的编码蛋白在调控昆虫精细胞发育中的应用。
本发明还提供了所述瓜实蝇TSSK1或TSSK3基因的编码蛋白在调控昆虫雄性生殖能力中的应用。
本发明还提供了所述瓜实蝇ZcTSSK1或ZcTSSK3基因在害虫防治中的应用。
本发明还提供了一种防治昆虫的方法,包括如下步骤,采用TSSK1或TSSK3基因的dsRNA递送至昆虫体内。
特别的,所述TSSK1基因的dsRNA的扩增引物如SEQ ID No.11和SEQ ID No.12所示。
特别的,所述TSSK3基因的dsRNA的扩增引物如SEQ ID No.13和SEQ ID No.14所示。
具体的,所述昆虫为瓜实蝇。
进一步的,所述瓜实蝇为雄性。
本发明的有益效果:本发明的瓜实蝇的TSSK1和TSSK3基因时空和组织表达谱表明,这两个基因都在雄虫的精巢中特异性高表达,特别是在瓜实蝇成虫精巢组织中的转化区,信号富集在成熟精子细胞中,该区域精细胞发生形态分化,转化为有尾部的精子,参与调控精细胞形态分化。TSSK1和TSSK3基因,在参与维持瓜实蝇雄性生殖过程中发挥重要功能,当抑制瓜实蝇雄虫TSSK1和TSSK3基因时,都会造成瓜实蝇雄虫精子数量减少,降低瓜实蝇雄虫生殖力,并且将处理的雄虫与正常的雌虫交配,雌虫的产卵率与卵孵化率降低。这两个基因有望成为SIT潜在靶标应用于害虫防治,为瓜实蝇绿色防控提供新思路。
附图说明
图1.瓜实蝇TSSK1(A)和TSSK3(B)在不同组织表达模式,fFB,fMG,fMT,和fOV代表雌虫的脂肪体(fat body)、中肠(midgut)、马氏管(Malpighian tubule)和卵巢(ovary);mFB,mMG,mMT,和mTE代表雄虫的脂肪体、中肠、马氏管和精巢(testis);柱状图表示基因表达量平均值±标准误(SE),图中柱子上的不同字母表示显著性差异(P<0.05,one-wayANOVA,LSD)。
图2.瓜实蝇TSSK1(A)和TSSK3(B)在不同发育阶段表达模式,E:卵;L 1-7:1-7日龄幼虫;P 1-9:1-9日龄蛹;M1-9:1-9日龄雄成虫;F 1:1日龄雌成虫;F 5:5日龄雌成虫;F 1-9:1-9日龄雌成虫;柱状图表示基因表达量平均值±标准误(SE),图中柱子上的不同字母表示显著性差异(P<0.05,one-way ANOVA,LSD)。
图3.TSSK1和TSSK3在瓜实蝇精巢中的定位。(A)瓜实蝇精巢中的阴性对照FISH信号。(B)精巢样品中TSSK1的荧光信号。(C)精巢样品中TSSK3的荧光信号。刻度尺标在左下角。
图4.采用饲喂进行dsRNA递送,9天后TSSK1和TSSK3基因沉默效率检测,柱状图表示基因表达量平均值±标准误(SE),ns表示无显著性差异,星号表示差异显著(*P<0.05;**P<0.01;***P<0.001)。
图5.RNAi后瓜实蝇精巢精子数量统计,柱状图表示基因表达量平均值±标准误(SE),ns表示无显著性差异,星号表示差异显著(*P<0.05;**P<0.01;***P<0.001)。
图6.各个处理的精子荧光信号。(A)dsGFP阴性对照处理后的瓜实蝇的精子荧光信号。(B)dsTSSK1处理后的瓜实蝇的精子荧光信号。(C)dsTSSK3处理后的瓜实蝇的精子荧光信号。刻度尺标在左下角。
图7.干扰TSSK1(A)和TSSK3(B)对瓜实蝇产卵量的影响,柱状图表示基因表达量平均值±标准误(SE),ns表示无显著性差异,星号表示差异显著(*P<0.05;**P<0.01;***P<0.001)。
图8.干扰TSSK1(A)和TSSK3(B)对瓜实蝇卵孵化率的影响,柱状图表示基因表达量平均值±标准误(SE),ns表示无显著性差异,星号表示差异显著(*P<0.05;**P<0.01;***P<0.001)。
具体实施方式
实施例1瓜实蝇TSSKs基因开放阅读框的获得
利用NCBI Primer BLAST(http://www.ncbi.nlm.nih.gov/tools/primer-blast)在线网站设计全长克隆PCR特异性引物,上下游引物序列如表1。PCR扩增条件如下:预变性:98℃反应3min,随后,98℃变性10s,55℃退火30s,72℃延伸1min,循环35次,最后72℃延伸10min。25μL的反应体系包含9.5μL无核酶水,12.5μL 2×PrimeSTAR Max Premix(TaKaRa,Japan),上下游引物各1μL(10μM),1μL瓜实蝇成虫精巢cDNA为模板。
PCR扩增产物经1%琼脂糖凝胶电泳检测,回收目的条带。然后连接到T-Easy vector上4℃过夜,具体操作见DNA连接试剂盒(TaKaRa,Japan)。并将5μL连接产物加入到50μL的Trans5α感受态细胞中,混匀,置冰上冰浴30min。42℃水浴热激65s,再放入冰浴中5min。加入无氨苄抗性的200μL LB液体培养基,37℃,250rpm振荡培养1h。在已加入氨苄的LB固体培养基上,将活化好的菌液80μL均匀涂布在上述LB平板上,37℃培养过夜,挑取白色正圆型的菌斑菌落37℃摇菌培养3~5h,菌液PCR检测,将阳性单克隆菌液送至华大基因科技有限公司测序。测序结果用DNAMAN软件和原序列比对验证。
表1.用于克隆、qRT-PCR和dsRNA合成所用到的引物对
瓜实蝇TSSK1开放阅读框序列,SEQ ID No.1
GCAAATTCAAATCATCAGAGTACCATCTTCAGATGCAATTAGACAAAATTTCAACATTATAAAAATCCA
ACAATTATGTCAAAATTTTCGACAACCTCCAATCGCCAACTGAATACGCGCAGCTCCGACATCGATGC
CCTGGCACAGCGTGGCTACAATATCGGACACAAAATTGGCGAGGGTTCCTATGCGACCGTCATCACCG
CTGGCTATGCAGACGATGCGGGTCATGGTGTACACTTGGCCTGCAAGATAATTGACAAGGCCAAGGC
ACCCTCGGATTTCGTACACAAATTCTTTCCACGTGAATTGGAAATTCTAACAAAAATCGATCATCCAAA
CATCATACAGATACACAGTATACTACAGCGTGGACCGAAGATATTCATATTCATGCGTTATGCGGAGAA
TGGTGATCTATTGAGTCATATCAAGAAATCGGGACCGGTTGAGGAGTTGCAAGCGAAAGCCTGGTTCA
TGCAGATGGCAAAGGCGCTCAAGTATCTGCATTCGCACGATATCGCCCATCGCGATCTGAAGTGCGAG
AACATATTGCTTTCGAAACGTCTCAATATCAAATTGGCTGATTTCGGCTTTGCACGCTATTGCTGCGAC
GATAGTGGACGTGAAATTAAATCGGAAACCTATTGCGGTTCGGCGGCCTATGCGGCACCTGAAGTCGT
ATGCGGTCGGCCATACGATCCGAAATTGGCCGACGCTTGGTCACTTGGCGTTATACTATTCATAATGTT
GAATGCGAAAATGCCGTTCGACGATAGCAACCTAAGCAAGTTGCTGGACGATCAGCGCAATAAGAAG
TTCGCATTTCGACGGAAATTGCAGGATCACATTTCGGCACAGGCCAAGGCCACCGTGGCGGTACTGCT
GGAGCCGGAACCGCATGCGCGTTGGAATTTACGTGAAATATTGAATTGCAGCTGGTTGCGTGCCGACG
AGGAGGAACCGTTGGCTTAGTCTAAGACGAGTACGTGCCTCATCCAACATGCAACATTTTGAGCAGCA
TGTCTGATTGGTTTTGGACCTAATGCCGTTGAATTTTTGTATGGGAAAAAAAGTAATTTCTAATATCTAT
ATATATTTTTTTGATTTGCGCATGTAAAATGTTGGTCATTCGTGAAATTGCCCTCTAAACACACTGCAAA
GCCTGTATATACACAACTTTTGGTACTAAAATTATTATATATATATACACGCATGTATATATAATGCGCATT
TGTATATTATATATGTTTATATATAATATATCTATGCTTTATTTTGTTCG
瓜实蝇TSSK3开放阅读框序列,SEQ ID No.2
GCAAATCACAGAAGAATCCAAAAACAATTACCGTTGAAAAGTTAAAAGTACGGCGTAGTAGTGTGTA
GGCGATCCCATTCGGTTATGCCAATACCACCGCAAAAAACGTTCACGAAATGTGCGACGAGTAAAATT
GTAGAAAACCAAATCATCGACCATATCAATAATAATACGAACAAGACCGACGAAAGCACAAATGTTAA
CGAGGTGTTACGACAGCATGGTGGTGCTTCCAAAGAAGATGACCGATCGCAAGCAGACCTCTCCGTG
CAACGCGTAGGATCGGGTGCAACTTTAGCGACTGAAACAAAGTCCTACATAAATGGACGACCGAAAA
CCATATTAGAAGATCATGGCATAGTACTTGGTAAGGTGATAGGTACCGGTAATTATGCAAAGGTAAAAA
TTGGGTTTTCGGAGGAGTATGGCAAACGTGTCGCCGTTAAAATTATATCGAAAGTGAAAGCGCCAGCG
GAGTATACGACAAAATTTCTACCACGTGAAATTGAAGCCGTCAAGGGATTGCATCACGAGAACTTGAT
AACGTTCTATCAAAGTATCGAGACTAGTCATAGAGTTTATTTAATCATGCAACTGGCAGAGAATGGCAC
ACTACTCGACTATGTACGTGAAAAGAAGTTCCTTGAAGAGCCACATAGTCGCAATCTATTCCAACAAT
TGATCAGCGCCGTAGAATATATACATTCGAAGAATGTGGTGCATCGCGACATAAAATGCGAGAACCTC
CTACTAGATGAAACATATACGCTAAAACTAATCGACTTCGGTTTTGCGCGCAAAGATACGCGTACCAGT
GATCAGCAAGTGATACTCTCGAAAACCTTTTGCGGTAGCTATGCCTATGCGAGTCCTGAAATCCTCAA
AGGTATTGCCTACGATCCTTTCATGTCCGATGTCTGGGCATGTGGTGTTGTCTGTTATGCAATGGTTTTC
GGGAGACTACCCTACGATGGCTCCAATGTGCATATACTACTTAAACGCATCAATGCTGCACTTGCTTTT
CCTAAGAATCCGGTGGTCTCTTTTGAATGTAAACAGCTAATTGGCCACATATTGGCACCATTAAAAGTG
CGGTATGCGATACCGCAGATTAAAGAGGATTCCTGGTTTGGTAGAACCTAACGAATATGCTTACAAATG
AAAAAAATTTCTTTAAAACATTACACGGAAATTAAACTACAAATTCTAGTTTGCAATGAAATTGATTGT
TGATTTCCTAAG
实施例2瓜实蝇TSSKs的组织和不同发育阶段谱
采用qRT-PCR技术检测TSSK1和TSSK3基因在瓜实蝇雌雄成虫组织的相对表达量,包括雌雄虫的中肠、马氏管、脂肪体和精巢组织,以及瓜实蝇不同发育阶段的相对表达量,包括卵(2h),幼虫(1、3和7天)、蛹(1、5和9日龄)和雌雄虫(1、5和9日龄)。qRT-PCR特异性引物序列见表1,其中以αTub和βTub1作为评估ZcTSSK1和ZcTSSK3不同组织表达的内参基因,以rpl13和rps3作为评估TSSK1和TSSK3不同发育阶段表达的内参基因。
图1是瓜实蝇TSSK1(图1A)和TSSK3(图1B)在雌雄虫不同组织(中肠、马氏管、脂肪体、精巢)的相对表达量,图2是瓜实蝇TSSK1(图2A)和TSSK3(图2B)在雌雄虫不同发育阶段(2h卵、1,3,7日龄幼虫、1,5,9日龄蛹、1,5,9日龄雌雄虫)的相对表达量,误差线为3个生物重复平均值的标准误,柱子上不同的字母表示表达量差异显著性(p<0.05,one-way ANOVA,LSD)。从图1中可以看出瓜实蝇TSSK1和TSSK3基因在成虫精巢中高表达,在其它组织中基本不表达,在图2中可以看出瓜实蝇TSSK1和TSSK3基因的表达高峰出现在雄性成虫期,在雌成虫中不表达。
实施例3瓜实蝇TSSKs的精巢组织定位
利用表2所示核酸序列合成荧光探针进行原位杂交分析。首先通过在1x PBS缓冲液中解剖2~5日龄瓜实蝇雄成虫精巢,置于4%多聚甲醛溶液中4℃固定过夜;然后用2%PBST(内含TritonX-100)清洗3次,每次5min,并用0.25%盐酸溶液浸泡30min以降低背景后用2%PBST清洗3次,每次5min;再用30μg/mL蛋白酶K室温通透20min后用2% PBST再清洗3次,每次5min;接下来用4%多聚甲醛室温二次固定20min,再用2% PBST清洗3次,每次5min;然后样本在68℃下与探针(500倍稀释)孵育72h,并用2%PBST进行最后三次洗涤,每次15min,之后使用0.5μg/mL 4’6’-二氨基-2-苯基吲哚(DAPI)(Sigma,USA)对样品染色10分钟,并使用LSM780激光共聚焦显微镜(Leica,USA)拍摄图像。
图3显示TSSK1(图3A)和TSSK3(图3B)在瓜实蝇精巢组织中的定位,结果表明目标信号出现在转化区,信号主要富集在成熟精子细胞中。所表达区域与精巢的输精管相连,为成熟精子的转化与形成区域。推测2个目的基因可能是通过参与精细胞形态转化来行使其生理功能。
表2.TSSK1和TSSK3原位杂交探针序列信息
实施例4TSSK1和TSSK3基因的dsRNA的制备
以上述TSSK1和TSSK3的开放阅读框序列为依据,利用NCBI Primer BLAST(http://www.ncbi.nlm.nih.gov/tools/primer-blast)在线网站,设计dsRNA引物,并在引物序列5’端加上一个T7启动子序列(TAATACGACTCACTATAGGG),序列信息如表1。
以瓜实蝇5日龄精巢组织cDNA为模板,利用基因dsRNA特异性引物普通PCR扩增TSSK1和TSSK3基因部分序列,阳性克隆,送往华大基因测序,测序无误后,以各自菌液为模板,用dsRNA引物完成PCR扩增,反应体系及条件同分子克隆。回收及纯化PCR产物,以较高浓度的PCR产物为模板,参照Transcript Aid T7 High Yield Transcription Kit(Thermo,USA)试剂盒说明书,合成和纯化dsRNA。然后用1.2%琼脂糖凝胶电泳检测dsRNA的纯度和完整性,并用紫外分光光度计(Thermo,USA)在260nm下测量其浓度,然后将其保持在-80℃下备用。
实施例5分别饲喂TSSK1和TSSK3基因片段合成的dsTSSK1、dsTSSK3来抑制瓜实蝇雄性生殖能力实验
(1)饲喂TSSKs基因片段合成的dsTSSK1和dsTSSK3
收集同一天羽化的雌、雄虫单性饲养,每个养虫笼中放置10头雄虫以饲喂dsRNA,对照饲喂dsGFP。从第1天以每头虫每天2μg/fly开始饲喂,晚上22点取出饲料,早上9点将20μg的dsRNA混合100μL液体饲料装入1.5mL无核酶离心管盖子中,移入养虫笼中,并将其与红色染料混合以观察试虫取食情况;下午14点成虫饲料正常饲喂。每天均重复此操作,连续饲喂至第9天。
(2)TSSKs基因沉默效率的检测
将连续饲喂9天的瓜实蝇雄成虫采集5只,使用TRIzol(Invitrogen,USA)试剂提取总RNA,使用RQ1 RNase-Free DNase试剂(Promeg,USA)和PrimeScriptTM RT Reagent Kit(TaKaRa,Japan)反转录获得模板cDNA,以此为模板进行qRT-PCR检测TSSK1和TSSK3基因的相对表达量,内参基因为αTub和rps3,用上述引物和方法进行qRT-PCR,以此来计算目的基因的沉默效率。图4结果显示,连续饲喂dsRNA 9天后,TSSK1和TSSK3表达较对照比分别显著下调68%和76%,说明使用饲喂的方式能够有效沉默靶基因的表达。但由于两个家族基因之间有一定的同源性,所以在饲喂ZcTSSK3时亦对TSSK1的表达量产生了一定的影响。
(3)通过精子数量以及产卵量和卵孵化率的变化来观察瓜实蝇雄性生殖能力的变化
在连续饲喂dsRNA后,通过在1×PBS中解剖9日龄成虫的精巢组织,用镊子撕开精巢表皮,将其置于100μL 1×PBS液中漂洗,DAPI原液稀释2000倍,10μL DAPI稀释液与10μL精子混合染色,室温下避光染色15min,取10μL混合液制片观察,激光共聚焦显微镜下拍照并计数精子数量,每个样品计数3次重复。处理后的雄虫与正常饲喂的同日龄雌虫交配,对照设置为饲喂dsGFP的雄虫与正常饲喂雌虫交配,交配结束后单对置于养虫笼(30×30×40cm)中过夜,第2天将雄虫取出,将10头交配过的雌虫放于同一养虫笼中,统计其连续5天内的产卵量和卵孵化率,设置三个生物重复。
图5和图6为dsTSSK1、dsTSSK3和dsGFP处理后的瓜实蝇成虫的精子数量统计。结果显示,dsTSSK1和dsTSSK3处理的雄虫精子数量与对照组相比,分别显著减少40%和53%。图7为dsRNA连续饲喂9天后雄虫与正常饲养的雌成虫交配,连续5天内的产卵数量,结果显示靶基因dsRNA处理组的产卵数量与对照组相比没有显著性差异。图8显示dsRNA连续饲喂9天后雄虫与正常饲喂的雌虫交配,连续5天内的卵孵化率,结果显示靶基因dsRNA饲喂组的卵孵化率相比对照组显著降低。
Claims (10)
1.瓜实蝇TSSK1基因,其特征在于,其核苷酸序列如SEQ ID No.1所示。
2.瓜实蝇TSSK3基因,其特征在于,其核苷酸序列如SEQ ID No.2所示。
3.权利要求1或2所述瓜实蝇TSSK1或TSSK3基因的编码蛋白在调控昆虫精细胞发育中的应用。
4.权利要求1或2所述瓜实蝇TSSK1或TSSK3基因的编码蛋白在调控昆虫雄性生殖能力中的应用。
5.权利要求1或2所述瓜实蝇TSSK1或TSSK3基因在害虫防治中的应用。
6.一种防治昆虫的方法,其特征在于,采用权利要求1或2所述TSSK1或TSSK3基因的dsRNA递送至昆虫体内。
7.如权利要求6所述方法,其特征在于,所述TSSK1基因的dsRNA的扩增引物如SEQ IDNo.11和SEQ ID No.12所示。
8.如权利要求6所述方法,其特征在于,所述TSSK3基因的dsRNA的扩增引物如SEQ IDNo.13和SEQ ID No.14所示。
9.如权利要求3~8任一项所述应用或方法,其特征在于,所述昆虫为瓜实蝇。
10.如权利要求9所述应用或方法,其特征在于,所述瓜实蝇为雄性。
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