CN116769819A - 柑橘CsWRKY40基因在驱避柑橘木虱中的应用 - Google Patents
柑橘CsWRKY40基因在驱避柑橘木虱中的应用 Download PDFInfo
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Abstract
本发明公开了柑橘CsWRKY40基因在驱避柑橘木虱中的应用。所述CsWRKY40基因的核苷酸序列如SEQ ID NO.1所示。本发明发现柑橘木虱对CsWRKY40‑VIGS的沉默柠檬材料具有很强的趋向性,进而说明CsWRKY40是一个具有驱避柑橘木虱能力的基因,因此该基因可以作为培育抗木虱柑橘品种的重要候选基因。本发明可为培育抗木虱柑橘品种提供重要的基因资源和有力的理论支撑。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及柑橘CsWRKY40基因在驱避柑橘木虱中的应用。
背景技术
柑橘是非常重要的经济作物,在全世界分布广泛。柑橘黄龙病(CitrusHuanglongbing,HLB)是当前全球柑橘生产上的头号病害,主要分布在亚洲、非洲、大洋洲、南美洲和北美洲的近50个国家和地区,给全世界柑橘产业造成巨大的危害。黄龙病的病原菌是一种寄生在柑橘植物韧皮部的革兰氏阴性细菌,目前该病原菌不能体外纯培养,同时尚未发现完全抗黄龙病的种质资源和黄龙病特异诱导的主效抗性基因,因此严重制约了抗病育种进程。柑橘木虱是黄龙病菌田间传播的媒介昆虫,它的发生会导致黄龙病的严重扩散。目前在柑橘生产中对木虱的防治方法主要集中在物理、化学和生物防治等方面,然而这些方法无论是实用性还是经济性尚不理想,因此培育抗木虱柑橘品种被认为是防治木虱的最优方案。
申请者在前期研究中利用RNA-Seq技术,以柑橘品种----甜橙为材料,对被柑橘木虱取食前后的甜橙叶片的全局转录组变化进行了评估,鉴定出在木虱取食前后的重要差异表达基因。通过挖掘柑橘中驱避木虱的相关功能基因,明确其对木虱的行为调控,可为培育抗木虱柑橘品种提供重要的基因资源和有力的理论支撑。
发明内容
本发明的目的在于克服现有技术的不足,提供了一种柑橘CsWRKY40基因及其在驱避柑橘木虱中的应用。本发明从柑橘品种甜橙(Citrus sinensis)中鉴定到一个转录因子CsWRKY40,该基因受柑橘木虱取食强烈诱导表达上调,通过利用病毒诱导的基因沉默技术(virus induced gene silencing,VIGS)抑制CsWRKY40基因的表达,从而使其表现出CsWRKY40基因功能丧失或表达水平下降的表型。结果发现:相比对照柠檬材料,柑橘木虱对CsWRKY40-VIGS的沉默柠檬材料具有很强的趋向性,由此说明CsWRKY40是一个具有驱避柑橘木虱能力的基因。该研究结果揭示CsWRKY40能够参与柑橘木虱诱导的防御反应,可以作为培育抗木虱柑橘品种的候选基因。
因此,本发明的第一个目的是提供柑橘CsWRKY40基因在驱避柑橘木虱中的应用,所述CsWRKY40基因的核苷酸序列如SEQ ID NO.1所示。
上述CsWRKY40基因编码蛋白的氨基酸序列如SEQ ID NO.2所示。应当理解,在不影响上述的CsWRKY40转录因子结构和活性的前提下,本领域技术人员可对SEQ ID NO.2所示的氨基酸序列进行各种取代、添加和(或)缺失一个或几个氨基酸获得具有同等功能的蛋白。
本发明的第二个目的是提供柑橘CsWRKY40基因在植物育种中的应用,所述CsWRKY40基因的核苷酸序列如SEQ ID NO.1所示。优选的,是在植物抗木虱育种中的应用。优选的,所述的植物为柑橘属植物。
本发明的第三个目的是提供柑橘CsWRKY40基因在植物种质资源改良中的应用,所述CsWRKY40基因的核苷酸序列如SEQ ID NO.1所示。优选的,是在提高植物抗虫性的种质资源改良中的应用。优选的,所述的植物为柑橘属植物。
本发明通过反向验证实验证明,抑制柠檬材料CsWRKY40基因的表达,会增加柑橘木虱对柠檬材料的趋向性,据此,本发明推断,过量表达CsWRKY40基因可以降低柑橘木虱对柠檬材料的趋向性。
因此,本发明的第四个目的是提供一种降低柑橘木虱对植物趋向性的方法,通过转基因技术过量表达柑橘CsWRKY40基因,所述的CsWRKY40基因的核苷酸序列如SEQ IDNO.1所示。优选的,所述的植物为柑橘属植物。
本发明公开了柑橘品种甜橙的CsWRKY40基因在柑橘抗木虱育种中的应用。本发明成功克隆了甜橙的CsWRKY40基因,并发现柑橘木虱对CsWRKY40-VIGS的沉默柠檬材料具有很强的趋向性,进而说明CsWRKY40是一个具有驱避柑橘木虱能力的基因,因此该基因可以作为培育抗木虱柑橘品种的重要候选基因。本发明可为培育抗木虱柑橘品种提供重要的基因资源和有力的理论支撑。
附图说明
图1是CsWRKY40基因对柑橘木虱取食的应答结果。
图2是柑橘木虱对CsWRKY40-VIGS的沉默柠檬材料的趋向性实验结果。
具体实施方式
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1:获得甜橙CsWRKY40基因
本发明利用生物信息方法结合PCR扩增的方法克隆基因。具体是在甜橙基因组数据库(http://citrus.hzau.edu.cn/index.php)中进行查找,查询到CsWRKY40的基因ID是Cs7g06330。以甜橙叶片的cDNA为模板,根据CsWRKY40的序列设计上游引物:
F:ATGGATTCTCACAACTCTC;和下游引物R:TCACATTGGTCTATGTGG,进行PCR产物扩增,扩增得到CsWRKY40基因的编码序列cDNA片段(核苷酸序列如SEQ ID NO.1所示),其长度为777bp。该基因编码258个氨基酸,氨基酸序列如SEQ ID NO.2所示。
CsWRKY40基因的cDNA序列如SEQ ID NO.1所示,具体为:
ATGGATTCTCACAACTCTCCTAAAGAAAAGATGAAATTGCTGCAAGCGAAATTGGAACATGTTCGC
AAACAGAATGAGAATCTGAGGCATCTGGTGAAAGCAATGAATAATCAATGCAATGATCTCCTGGCA
CGTATCCACGAAGCTAATAGAACATATTCCTCCTCTGATCATCATCACTTCAACAATAATATTAATATT
GGAGGAGTCACAGCACAAGTTCCTCCTGTTCCAAATGCCAAGCAATCAAGAATCTTTGTGAAAGCA
GATTCTAAGGACAGTAGCCTTATCGTGAAAGATGGACATCAATGGAGAAAGTACGGCCAAAAGGTT
ACTAAAGATAATCCCTCACCTCGGGCTTATTTCAGATGTTCAATGGCTTCTTCTGGATGTCCTGTAAA
AAAAAAGGTTCAAAGATGCATGGAGGACAAGTCATTTCTCGTTGCAACTTATGAAGGAGAACACA
ACCATGATGTTCAATGTAGCTCACTTGGGCAATCCTCCTCTTTAACAAATTATTGCTCACCCAAAAGC
TCAATTGTACATTGCCCAGATTATCAAACAACTGATTCTTTTGGATCAGATGTCACACTTGATCTTAC
TCTATCTGGGTCCAATCAAGAAACTAGACCCCCTAGAAACTTGATGCAAGTCTGTGACGATAAAAA
GAAGATTGAAGAATATGTGGCATCCTTAACTAAAGATCCCAGCTTCACCATAGCTGTAGCCGATGCTGTTGCAAGTTCCATCAATGGCCCCCCACATAGACCAATGTGA。
CsWRKY40基因编码蛋白的氨基酸序列如SEQ ID NO.2所示,具体为:MDSHNSPKEKMKLLQAKLEHVRKQNENLRHLVKAMNNQCNDLLARIHEANRTYSSSDHHHFNNNINIGGVTAQVPPVPNAKQSRIFVKADSKDSSLIVKDGHQWRKYGQKVTKDNPSPRAYFRCSMASSGCPVKKKVQRCMEDKSFLVATYEGEHNHDVQCSSLGQSSSLTNYCSPKSSIVHCPDYQTTDSFGSDVTLDLTLSGSNQETRPPRNLMQVCDDKKKIEEYVASLTKDPSFTIAVADAVASSINGPPHRPM*。
实施例2:CsWRKY40基因的表达受柑橘木虱取食强烈诱导
在柑橘木虱取食甜橙幼苗实验中,先将柑橘木虱成虫饥饿处理12h,然后将其放入含有3个月苗龄甜橙幼苗的玻璃装置中,分别收集木虱取食12、24、48h的甜橙叶片,0h作为对照。提取叶片RNA,利用荧光定量PCR技术,检测CsWRKY40基因的表达情况。CsWRKY40定量引物对如下所示:CsWRKY40-qRT-F:CTGTTCCAAATGCCAAGCA;CsWRKY40-qRT-R:CCCGAGGTGAGGGATTATCT。
结果发现:CsWRKY40基因的表达受柑橘木虱取食强烈诱导上调(图1),说明CsWRKY40基因可能参与柑橘木虱诱导的防御反应。
实施例3:沉默CsWRKY40基因的柠檬增强了柑橘木虱的趋向性
(1)载体构建
由于CsWRKY40基因在甜橙和柠檬中的序列相同,我们以柠檬叶片cDNA为模板,设计引物在柠檬中扩增CsWRKY40基因的特异300bp片段,通过同源重组连接到pTRV2载体的Xba I及Sma I两个酶切位点中间,引物设计如下:
CsWRKY40-TRV-F:AAGGTTACCGAATTCTCTAGAAGATGAAATTGCTGCAAGC;
CsWRKY40-TRV-R:TGTCTTCGGGACATGCCCGGGTTTGGCCGTACTTTCTCCATT;
扩增体系:50μL体系,上下游引物各2μL,cDNA1μL,2×Phanta Max Master Mix(Dye Plus)25μL,H2O 20μL。载体测序正确后提取质粒,转化入农杆菌感受态GV3101中。
分别将质粒pTRV1和pTRV2转化入农杆菌感受态GV3101中,得到TRV1农杆菌和TRV2农杆菌。
(2)农杆菌侵染液准备
分别挑取TRV1,TRV2与CsWRKY40-pTRV2农杆菌单克隆在5mL含有对应抗生素的LB液体培养基内振荡活化过夜(50mg/L卡那霉素,50mg/L利福平)。按照1:100的体积比将活化完成的农杆菌菌液接种到含有抗生素的新鲜LB培养基中,28℃培养过夜。4000r/min离心10min,收集菌体,加入侵染缓冲液(10mmol/L MES,10mmol/L MgCl2,200μmol/L AS,pH=5.6~5.7)悬浮菌体并调节OD600至1.0。按照1:1的体积比混合TRV1与TRV2或CsWRKY40-pTRV2菌体重悬液。黑暗处静置2-3h后即可用于侵染。
(3)农杆菌转化柠檬种子
将新鲜的柠檬种子从果实中完整取出,用1mol/LNaOH溶液浸泡15min去除果胶,后用灭菌水冲洗干净,平铺于干净纱布上,放置于30℃培养箱内催芽,待种子幼芽萌发至1-2cm长时即可用于VIGS侵染。将萌发的柠檬种子浸泡于配置好的侵染液中,真空抽气装置抽真空1min,快速放气,再静置10min,此步骤可重复2-3次。将种子捞出,在滤纸上晾干菌液,后放入铺有无菌水浸湿滤纸的皿中,暗室放置3d后种在土中,待其长至合适大小即可进行阳性鉴定。
(4)阳性苗鉴定及柑橘木虱趋向性实验
以提取出的柠檬DNA为模板,采用两对引物鉴定阳性植株,TRV1正反引物,TRV2正向引物和CsWRKY40-pTRV2反向引物。引物序列如下:
TRV1-F:TTACAGGTTATTTGGGCTAG;
TRV1-R:CCGGGTTCAATTCCTTATC;
TRV2-F:TGGGAGATGATACGCTGTT;
V2-40-R:TGTCTTCGGGACATGCCCGGGTTTGGCCGTACTTTCTCCATT;
同时对获得的CsWRKY40-VIGS的沉默柠檬材料的CsWRKY40基因表达进行分析,CsWRKY40定量引物为:CsWRKY40-qRT-F:CTGTTCCAAATGCCAAGCA;CsWRKY40-qRT-R:CCCGAGGTGAGGGATTATCT。
对获得的CsWRKY40-VIGS的3个沉默柠檬株系(CsWRKY40-pTRV2-21#、CsWRKY40-pTRV2-30#和CsWRKY40-pTRV2-32#)进行柑橘木虱趋向性实验。分别在2、4、8、12、24、48、72、96h统计沉默柠檬材料和对照材料(Control-pTRV2-#2、Control-pTRV2-#7和Control-pTRV2-#4)上的落虫数量。
结果表明:CsWRKY40-pTRV2-21#、CsWRKY40-pTRV2-30#和CsWRKY40-pTRV2-32#的CsWRKY40基因表达分别降低到对照的16.7%、3.3%和0.75%(图2a)。CsWRKY40-VIGS的沉默柠檬材料上的落虫数量明显多于对照材料(图2b),即柑橘木虱对CsWRKY40-VIGS的沉默柠檬材料具有很强的趋向性,由此说明CsWRKY40是一个具有驱避柑橘木虱能力的基因。
以上仅是本发明的优选实施方式,应当指出的是,上述优选实施方式不应视为对本发明的限制,本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说,在不脱离本发明的精神和范围内,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.柑橘CsWRKY40基因在驱避柑橘木虱中的应用,其特征在于,所述CsWRKY40基因的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用,其特征在于,所述CsWRKY40基因编码蛋白的氨基酸序列如SEQ ID NO.2所示。
3.柑橘CsWRKY40基因在植物育种中的应用,其特征在于,所述CsWRKY40基因的核苷酸序列如SEQ ID NO.1所示。
4.根据权利要求3所述的应用,其特征在于,是在植物抗木虱育种中的应用。
5.根据权利要求3或4所述的应用,其特征在于,所述的植物为柑橘属植物。
6.柑橘CsWRKY40基因在植物种质资源改良中的应用,其特征在于,所述CsWRKY40基因的核苷酸序列如SEQ ID NO.1所示。
7.根据权利要求6所述的应用,其特征在于,是在提高植物抗虫性的种质资源改良中的应用。
8.根据权利要求6或7所述的应用,其特征在于,所述的植物为柑橘属植物。
9.一种降低柑橘木虱对植物趋向性的方法,其特征在于,通过转基因技术过量表达柑橘CsWRKY40基因,所述的CsWRKY40基因的核苷酸序列如SEQ ID NO.1所示。
10.根据权利要求9所述的方法,其特征在于,所述的植物为柑橘属植物。
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