CN118006668A - 柑橘木虱DcS44基因在防治柑橘木虱及黄龙病中的应用 - Google Patents
柑橘木虱DcS44基因在防治柑橘木虱及黄龙病中的应用 Download PDFInfo
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Abstract
本发明提供一种柑橘木虱DcS44基因在防治柑橘木虱及黄龙病中的应用，所述柑橘木虱DcS44基因的核苷酸序列如SEQ ID NO.1所示。本发明设计特异性引物合成dsRNA，借助RNA干扰沉默DcS44基因，检测其基因沉默效率，发现DcS44dsRNA可使柑橘木虱DcS44基因相对表达量减少，提高柑橘木虱死亡率提高，从而达到控制柑橘木虱虫口数量防止黄龙病扩散成灾的目的。
Description
技术领域
本发明属于生物基因工程技术领域，具体涉及一种柑橘木虱DcS44基因在防治柑橘木虱及黄龙病中的应用。
背景技术
黄龙病(Huanglongbing,HLB)是当前全球柑橘产业最具有毁灭性的病害，又称柑橘“癌症”。目前，尚未发现抗HLB柑橘品种，且对感病树体缺乏有效的治疗方法，只砍掉病株减少传播菌源(Grafton-Cardwell et al.,2013；Alquézar et al.,2022)。我国HLB常年发生面积超过200万亩，其中江西、广东、广西、福建等柑橘产区面临着HLB的严重威胁(Fu etal.,2020；Zhou,2020)。柑橘木虱(Diaphorina citri Kuwayama)是传播黄龙病菌的最主要虫媒，而控制木虱虫口数量是防止黄龙病扩散成灾的关键措施之一。当前，柑橘木虱防控主要通过喷洒农药，而大规模和高频率使用农药造成了抗药性木虱、水土污染和药物残留等问题。因此，面对日益严重的柑橘黄龙病和环境问题，降低化学农药使用量，开发绿色无公害的柑橘木虱防控方法显得十分重要和迫切。
发明内容
鉴于此，本发明提供一种柑橘木虱DcS44基因在防治柑橘黄龙病中的应用。测定了DcS44基因沉默对木虱存活的影响，实验结果表明：DcS44基因沉默后，柑橘木虱的死亡率显著增加，对于柑橘木虱防治以及抗木虱植物的培育具有重大价值。
为达到上述目的，本发明采用以下技术方案：
本发明目的之一是提供一种柑橘木虱DcS44基因在防治柑橘木虱及黄龙病中的应用，所述柑橘木虱DcS44基因的核苷酸序列如SEQ ID NO.1所示，SEQ ID NO.1具体如下：
ATGGCGACTTTGTCCGCGAAAATCTTCATGTATATGTTTATCGTATATGTCGCGGTCTCTCAGGTGGAGTGCGAGCCACCAGCTCCAGCACCAACTGACGCCACCAAACGTGCAGAAAGTCCGGCCGAAGAGAAACTCAATCCAGCGGAGGTCGAAAAACAAAACAAAATCCTCGACTGTGTCTTCGAGAATGTTCAAAAGAAGTATCAGGTTAACGGTGTATCAAAGGAACAGTTTGACAAGAATATACGCTTGTCCGACGAGGACAGTATGAAGACTTTGTTCGAATCTGCAGGCTTTAAGGGAAATATTGGGGAATTGTTCGATTATATTGACTTCTCTTTTGAGGTGTGCTGGGCAAAGCCTCAGACTACAAACGGATCAGCTAATGCCCCCTCTTCATAG。
进一步地，所述应用通过DcS44基因沉默使柑橘木虱死亡从而减少传播来防治柑橘黄龙病。
进一步地，所述基因沉默为采用RNA干扰技术进行基因沉默。
进一步地，所述RNA干扰技术包括如下步骤：以柑橘木虱cDNA为模板，通过PCR技术，使用扩增引物获得DcS44基因的部分片段，扩增产物连接入质粒中，以质粒为模板，用含启动子的引物进行扩增，扩增产物胶回收，合成dsRNA；稀释合成的dsRNA，显微注射柑橘木虱。
进一步地，所述扩增引物如下：
DcS44 F:ATGGCGACTTTGTCCGCGA；
DcS44 R:CTATGAAGAGGGGGCATTAGCTG。
进一步地，所述含启动子的引物如下：
DcS44-T7 F:GGATCCTAATACGACTCACTATAGGGCCAGCTC CAGCACCAAC，
DcS44-T7 R:GGATCCTAATACGACTCACTATAGGGGTCTGA GGCTTTGCCCA。
进一步地，所述质粒为-Blunt。
本发明目的之二是提供一种生物杀虫制剂，所述生物杀虫制剂包含柑橘木虱DcS44基因的dsRNA。
进一步地，所述dsRNA一条链的核苷酸序列SEQ ID NO.2具体如下所示：
CCAGCTCCAGCACCAACTGACGCCACCAAACGTGCAGAAAGTCCGGCCGAAGAGAAACTCAATCCAGCGGAGGTCGAAAAACAAAACAAAATCCTCGACTGTGTCTTCGAGAATGTTCAAAAGAAGTATCAGGTTAACGGTGTATCAAAGGAACAGTTTGACAAGAATATACGCTTGTCCGACGAGGACAGTATGAAGACTTTGTTCGAATCTGCAGGCTTTAAGGGAAATATTGGGGAATTGTTCGATTATATTGACTTCTCTTTTGAGGTGTGCTGGGCAAAGCCTCAGAC。
本发明目的之三是提供一种柑橘木虱DcS44基因在培育抗柑橘木虱和抗黄龙病品种中的应用。
本发明的有益效果为：
筛选合适的靶标基因是RNAi抗虫的前提，本发明将DcS44 dsRNA导入木虱体内，测定了DcS44基因沉默对木虱存活的影响，实验结果表明DcS44基因沉默后，柑橘木虱的死亡率与对照GFP dsRNA相比显著增加，在注射DcS44 dsRNA 10天后柑橘木虱死亡率高达68％，对于柑橘木虱防治以及抗木虱植物的培育具有重大价值。
附图说明
图1为DcS44基因的ORF扩增产物凝胶电泳图。
图2为DcS44基因在柑橘木虱的各个发育时期表达量。
图3为DcS44基因在柑橘木虱的各个组织部位表达量。
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明，以使本领域的技术人员更加清楚地理解本发明。
关键试验材料来源及理化参数：
本次实验材料中的柑橘木虱，由赣南师范大学国家脐橙工程技术研究中心养虫室提供，饲养寄主是九里香；环境条件为温度(28±2)℃，湿度60％～80％，光周期L∶D＝14∶10h。
本发明中未对具体原料进行说明均为已经存在物质，可以从市面上直接购买得到。
实施例1
柑橘木虱DcS44基因的ORF扩增。
从饲养笼中用收集器收集100只柑橘木虱，在解剖镜下用镊子解剖出木虱的头部，然后利用Trizol提取柑橘木虱的RNA，利用One-Step gDNA Removal andcDNA Synthesis SuperMix反转录试剂盒(北京全式金生物技术股份有限公司)合成cDNA第一链。
根据柑橘木虱唾液蛋白转录组得到的DcS44基因序列，通过软件Premier Primer5.0设计特异性引物，命名为DcS44-F1和DcS44-R1。以cDNA作为模板，DcS44-F1和DcS44-R1为引物，用高保真酶PrimeSTAR Max Premix(Takara)扩增DcS44基因的ORF。
DcS44 F1:ATGGCGACTTTGTCCGCGA
DcS44 R1:CTATGAAGAGGGGGCATTAGCTG
将上述PCR产物进行电泳，结果如图1所示，其中，M：2000bp marker。
PCR的扩增产物Takara凝胶回收试剂盒进行回收纯化，与-Blunt克隆载体连接，转化大肠杆菌DH5α，测序鉴定。得到DcS44基因序列如下所示：
ATGGCGACTTTGTCCGCGAAAATCTTCATGTATATGTTTATCGTATATGTCGCGGTCTCTCAGGTGGAGTGCGAGCCACCAGCTCCAGCACCAACTGACGCCACCAAACGTGCAGAAAGTCCGGCCGAAGAGAAACTCAATCCAGCGGAGGTCGAAAAACAAAACAAAATCCTCGACTGTGTCTTCGAGAATGTTCAAAAGAAGTATCAGGTTAACGGTGTATCAAAGGAACAGTTTGACAAGAATATACGCTTGTCCGACGAGGACAGTATGAAGACTTTGTTCGAATCTGCAGGCTTTAAGGGAAATATTGGGGAATTGTTCGATTATATTGACTTCTCTTTTGAGGTGTGCTGGGCAAAGCCTCAGACTACAAACGGATCAGCTAATGCCCCCTCTTCATAG。
实施例2
柑橘木虱DcS44基因的表达分析。
为了研究DcS44基因在柑橘木虱不同组织部位和发育时期的表达情况，于是对柑橘木虱进行了解剖镜下的样本采集。解剖了雌雄柑橘木虱的头部、腿部、翅膀、表皮和中肠等柑橘木虱的不同组织部位，为了研究柑橘木虱DcS44基因在木虱不同发育阶段的表达水平，在解剖镜下采集了不同阶段的木虱样本，包括卵、一龄若虫、二龄若虫、三龄若虫、四龄若虫、五龄若虫和雌雄柑橘木虱成虫。同样采用Trizol RNA提取试剂盒上的操作提取柑橘木虱不同组织部位，以及不同发育时期的RNA。按照上述反转录试剂盒的说明进行操作，进行反转录合成cDNA。最后利用qRT-PCR的方法来检测柑橘木虱DcS44基因在不同组织部位和发育时期的的表达水平。所用引物为DcS44-F2和DcS44-R2，柑橘木虱的Actin基因(DcActinF和DcActin R)及GAPDH基因(DcGAPDH F和DcGAPDH R)作为qPCR内参，实时荧光定量所采用的试剂为Green qPCR SuperMix(北京全式金生物技术股份有限公司)。运用2-ΔΔCT(Ct表示循环数)法处理数据。检测结果见图2、3。
其中，qRT-PCR检测引物如下：
DcS44 F2:TCTGCAGGCTTTAAGGGAAATA，
DcS44 R2:CTGATCCGTTTGTAGTCTGAGG；
DcActin F:AGAAAGTACTCCGTGTGGATTG，
DcActin R:CGGACTCGTCGTATTCTTGTT；
DcGAPDH F:TGAGATCAAGGCCAAGGTAAAG，DcGAPDH R:GTCAAAGATGGAGGAGTGAGTG。
由图2、3可知：DcS44基因在柑橘木虱的各个发育时期均有表达,一龄和二龄若虫中表达量稍高，五龄的含量相对较低。DcS44基因在柑橘木虱不同部位的表达量存在很大的差异，其中表皮中的表达量最低，头部的表达量最高，如在雌虫头部的表达量是表皮的548倍，但两性木虱中同一组织部位的表达量差异不大，以上结果预示该基因可能在头部的唾液腺中高量表达。
实施例3
柑橘木虱DcS44基因的dsRNA合成。
1.引物设计与基因片段扩增
利用Primer Primer5.0软件设计引物DcS44 F3、DcS44 R3、DcS44-T7 F、DcS44-T7R，GFP-F、GFP-R、GDP-T7F、GFP-T7R，由上海生工公司合成。
其中，DcS44 dsRNA合成引物具体如下：
DcS44 F3:CCAGCTCCAGCACCAAC，
DcS44 R3:GTCTGAGGCTTTGCCCA；
DcS44-T7F:GGATCCTAATACGACTCACTATAGGGCCAGCTCCAGCACCAAC，
DcS44-T7R:GGATCCTAATACGACTCACTATAGGGGTCTGAGGCTTTGCCCA。
其中，GFP dsRNA合成引物具体如下：
GFP F:ACAAGTTCAGCGTGTCCG，
GFP R:TCACCTTGATGCCGTTCT；
GFP-T7 F:GGATCCTAATACGACTCACTATAGGGACAAGTTCAGCGTGTCCG，
GFP-T7 R:GGATCCTAATACGACTCACTATAGGGTCACCTTGATGCCGTTCT。
上述各引物序列中下划线处为T7 RNA聚合酶启动子序列。
PCR反应体系为：高保真酶PrimeSTAR Max Premix(2X)25μL、正向引物(10μmol·L-1)2μL、反向引物(10μmol·L-1)2μL、质粒2μL(稀释50倍)，ddH2O 19μL。
DcS44基因PCR的反应程序为：98℃10s，52℃15s，72℃30s；35个循环，4℃保存。
GFP基因PCR的反应程序为：98℃10s，56℃15s，72℃30s；35个循环，4℃保存。
以DcS44 ORF质粒为模板，用DcS44 F3和DcS44-T7 R、DcS44-T7F和DcS44 R3引物分别扩增，得到合成DcS44 dsRNA的DNA片段。
以16318hGFP质粒为模板，用GFP F和GFP-T7 R、GFP-T7 F和GFP R作为引物进行扩增，得到合成GFP dsRNA的DNA片段。
2.柑橘木虱DcS44基因和GFP的dsRNA的制备
将DcS44-F3和DcS44-T7 R、DcS44-T7 F和DcS44-R3扩增的2种DNA产物按照TaKaRaDNA产物纯化试剂盒说明书纯化。
将上述得到的2种纯化产物，测定浓度，作为体外转录dsRNA的模板。参考Promega公司的T7 RiboMAXTMExpress RNAi System试剂盒说明书进行以下步骤。dsRNA的体外转录体系为：
37℃孵育两小时后，将2个体系的产物混合。
dsRNA退火：70℃水浴10min，冷却至室温。
dsRNA纯化：加1μL(1:200无酶水稀释)RNAse、1μL RQ1，30℃孵育30min；加60μL无酶水、10μL醋酸钠、100μL异丙醇，冰上5min，12000rpm 10min；1mL 75％酒精清洗沉淀，7500rpm 5min；室温通风橱20min，加50μL无酶水溶解dsRNA。按此步骤得到柑橘木虱DcS44dsRNA，用相同步骤得到GFP dsRNA。
取1μL上述dsRNA，稀释10倍，剩余dsRNA置于-20℃冰箱保存；取2μL稀释后dsRNA用Nano Drop OneC超微量核酸仪测定浓度。
实施例4
dsRNA显微注射及生物检测。
柑橘木虱DcS44 dsRNA注射组：取10头羽化3天的柑橘木虱进行注射，每头注射500ng的DcS44 dsRNA，饲养在九里香植株。
柑橘木虱GFP dsRNA注射组：取10头羽化3天的柑橘木虱进行注射，每头注射500ng的GFP dsRNA，饲养在九里香植株。
上述各组设置10个重复，置于人工气候箱(温度(28±2)℃，湿度60％～80％，光周期L∶D＝14∶10)。每天统计各九里香中柑橘木虱的存活数，结果见表1。
表1 DcS44 dsRNA、GFP dsRNA注射组死亡率
| 组别 | GFP dsRNA注射组 | DcS44 dsRNA注射组 |
| Day1 | 0±0 | 0±0 |
| Day2 | 0±0 | 2±4.47 |
| Day3 | 4±8.94 | 14±11.40 |
| Day4 | 4±8.94 | 28±13.04＊ |
| Day5 | 8±10.95 | 36±15.17＊ |
| Day6 | 8±10.95 | 48±13.04＊ |
| Day7 | 8±10.95 | 50±12.25＊ |
| Day8 | 8±10.95 | 58±16.43＊ |
| Day9 | 8±10.95 | 58±16.43＊ |
| Day10 | 10±10 | 68±23.87＊ |
注：＊表示与对照组(GFP dsRNA)相比，试验组结果差异显著(t-test,n＝5，P<0.05)。
由表1可知：DcS44 dsRNA注射组死柑橘木虱的死亡率与对照GFP dsRNA相比显著增加，随着时间的推移死亡率呈上升趋势，在第十天时达到了68％的死亡率。
实施例5
DcS44 dsRNA抑制柑橘木虱DcS44基因的表达。
利用Primer Primer5.0软件设计扩增DcS44基因的引物DcS44-F2和DcS44-R2，内参基因Actin基因(DcActin F和DcActin R)及GAPDH基因(DcGAPDH F和DcGAPDH R)。
按实施例4收集各组dsRNA显微注射后存活48h及96h柑橘木虱样品，DcS44及GFPdsRNA各5组，每组5只柑橘木虱。用宝生物Trizol试剂盒提取柑橘木虱的RNA；用全式金反转录试剂盒反转录成cDNA；稀释20倍后作为实时荧光定量PCR的模板，DcS44-F2和DcS44-R2作为引物，Actin基因(DcActin F和DcActin R)及GAPDH基因(DcGAPDH F和DcGAPDH R)为内参基因。
实时荧光定量PCR体系为：正向引物(10μmol·L-1)0.4μL、反向引物(10μmol·L-1)0.4μL、2×Green qPCR SuperMix 10μL、模板cDNA 4μL，ddH2O 5.2μL。
PCR循环程序为95℃孵育10min；2步法95℃5s，60℃30s，40个循环；融解曲线95℃10s，65℃1min，97℃1s。每个样本3个重复，最终结果的计算采用2-△△Ct法(Ct表示循环数)进行计算，结果见表2。
表2不同注射组柑橘木虱DcS44基因相对表达量
| 组别 | GFP dsRNA注射组 | DcS44 dsRNA注射组 |
| Day2 | 1±0.16 | 0.14±0.11＊ |
| Day4 | 1±0.30 | 0.12±0.10＊ |
注：＊表示与对照组(GFP dsRNA)相比，试验组结果差异显著(t-test,n＝5，P<0.05)。
由表2可知：DcS44 dsRNA注射组柑橘木虱DcS44基因相对表达量与对照GFP dsRNA相比显著减少，随着时间的推移柑橘木虱DcS44基因相对表达量呈下降趋势。
由以上可知，DcS44 dsRNA可使柑橘木虱DcS44基因相对表达量减少，提高柑橘木虱死亡率提高，从而达到控制柑橘木虱虫口数量防止黄龙病扩散成灾的目的。
以上仅为本发明的较佳实施方案，并非用于限定本发明的保护范围。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.柑橘木虱DcS44基因在防治柑橘木虱及黄龙病中的应用，其特征在于，所述柑橘木虱DcS44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，所述应用通过DcS44基因沉默使柑橘木虱死亡从而减少传播来防治柑橘黄龙病。
3.根据权利要求2所述的应用，其特征在于，所述基因沉默为采用RNA干扰技术进行基因沉默。
4.根据权利要求3所述的应用，其特征在于，所述RNA干扰技术包括如下步骤：以柑橘木虱cDNA为模板，通过PCR技术，使用扩增引物获得DcS44基因的部分片段，扩增产物连接入质粒中，以质粒为模板，用含启动子的引物进行扩增，扩增产物胶回收，合成dsRNA；稀释合成的dsRNA，显微注射柑橘木虱。
5.根据权利要求4所述的应用，其特征在于，所述扩增引物如下：
DcS44 F:ATGGCGACTTTGTCCGCGA；
DcS44 R:CTATGAAGAGGGGGCATTAGCTG。
6.根据权利要求4所述的应用，其特征在于，所述含启动子的引物如下：
DcS44-T7 F:GGATCCTAATACGACTCACTATAGGGCCAGCTC CAGCACCAAC，
DcS44-T7 R:GGATCCTAATACGACTCACTATAGGGGTCTGA GGCTTTGCCCA。
7.根据权利要求4所述的应用，其特征在于，所述质粒为-Blunt。
8.一种生物杀虫制剂，其特征在于，所述生物杀虫制剂包含柑橘木虱DcS44基因的dsRNA。
9.根据权利要求8所述的生物杀虫剂，其特征在于，所述dsRNA核苷酸序列如SEQ IDNO.2所示。
10.柑橘木虱DcS44基因在培育抗柑橘木虱和抗黄龙病柑橘品种中的应用。
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