中国农业科学 ›› 2022, Vol. 55 ›› Issue (22): 4398-4407.doi: 10.3864/j.issn.0578-1752.2022.22.006
收稿日期:
2022-07-04
接受日期:
2022-07-16
出版日期:
2022-11-16
发布日期:
2022-12-14
通讯作者:
宋震
作者简介:
张琦,E-mail:基金资助:
ZHANG Qi(),DUAN Yu(
),SU Yue,JIANG QiQi,WANG ChunQing,BIN Yu,SONG Zhen(
)
Received:
2022-07-04
Accepted:
2022-07-16
Online:
2022-11-16
Published:
2022-12-14
Contact:
Zhen SONG
摘要:
【目的】 构建基于柑橘叶斑驳病毒(citrus leaf blotch virus,CLBV)的表达载体,通过系统表达抗菌肽提高植物抗病性,为柑橘溃疡病、柑橘黄龙病等病害的防控提供新型技术手段。【方法】 基于前期构建的侵染性克隆pCY-CLBV201,在外壳蛋白基因终止子后插入亚基因组启动子序列及多克隆位点,构建病毒表达载体pCLBV202。在多克隆位点插入绿色荧光蛋白(green fluorescent protein)基因(gfp),通过农杆菌介导接种、荧光观察验证pCLBV202-GFP表达GFP的情况。克隆天蚕的抗菌肽(cecropin B,CB)基因并构建重组载体pCLBV202-CB,通过农杆菌介导分别注射接种本氏烟和真空浸润接种柑橘实生苗,筛选阳性植株并分别注射接种和根灌接种烟草青枯病菌及针刺离体叶片接种柑橘溃疡病菌,同时设空载体接种植株为对照,通过症状观察、发病率及病情指数评价接种植株的烟草青枯病抗性;通过柑橘叶片的病斑数量、发病率及菌落浓度评价其溃疡病抗性。【结果】 pCLBV202-GFP接种烟草和尤力克柠檬后,均可以在系统新叶上观察到绿色荧光,在烟草上表现更为明亮,说明基于CLBV的表达载体构建成功。接种青枯病菌后,处理组(pCLBV202-CB)较对照组(pCLBV202)发病时间延迟4 d。在接种后第24天(24 dpi),处理组发病率为14.3%,对照组发病率为100%,差异显著。处理组相对于对照组的抗性指数为-2.66,抗性评价为高抗,表明利用pCLBV202-CB系统表达CB增强了对烟草青枯病的抗性。尤力克柠檬叶片针刺接种柑橘溃疡病菌,7 dpi时,处理组病斑数为47个,发病率为43.5%,对照组病斑个数为73个,发病率为67.6%。菌群数变化检测发现,处理组菌群数小于对照组菌群数,表明利用pCLBV202-CB系统表达CB增强了尤力克柠檬的溃疡病抗性。【结论】 构建了基于CLBV的病毒表达载体pCLBV202。利用pCLBV202在本氏烟和柑橘中系统表达CB可以提高植株对细菌性病害的抗性,这为柑橘细菌性病害的防控提供了新技术。
张琦,段玉,苏越,蒋琪琪,王春庆,宾羽,宋震. 基于柑橘叶斑驳病毒的表达载体构建及应用[J]. 中国农业科学, 2022, 55(22): 4398-4407.
ZHANG Qi,DUAN Yu,SU Yue,JIANG QiQi,WANG ChunQing,BIN Yu,SONG Zhen. Construction and Application of Expression Vector Based on Citrus Leaf Blotch Virus[J]. Scientia Agricultura Sinica, 2022, 55(22): 4398-4407.
表1
试验所用引物序列"
引物rimer | 引物序列Primer sequence (5′-3′) |
CLBV1F | AGCCATAGTTGAACCATTCCTC |
CLBV5R | GCAGATCATTCACCACATGC |
(3+1)F | AACAAAGCAAAACTCTTAGAAATGTAGCCC |
(3+1)R | CTTTAAGAGTGTAAAGTCCTGGCCCACCCA |
5-F | TAGATACTAGCAGAACTCGAGCCGAAACAGCTGTTATT |
5-R | ACACATAATCCTTCACTACATTTCTAAGAGTTTTGC |
3-F | GAGCTGTACAAGTGACCCGGGTCCCGAATTCTGGCATGGGC |
3-R | AAATGTTTGAACGATATCGGGGAAATTCGAGCTCT |
92bpf1 | CAAAACTCTTAGAAATGTAGGAAGGATTATGTGTCTCATGTTAAGTCAG |
92bpr1 | GAGATTGGGGTAATGATAGATTGAGAAAATCCCGGGATGAGTAAAGGAGAAGAACTTTTCAC |
gfpf1 | GTGAAAAGTTCTTCTCCTTTACTCATCCCGGGATTTTCTCAATCTATCATTACCCCAATCTC |
gfpr1 | GCCCATGCCAGAATTCGGGATCCCCCGGGTTATTTGTATAGTTCATCCATGCCATGT |
图2
pCLBV202-GFP与pCLBV202-CB表达载体的构建 A:pCLBV202-GFP单克隆检测 pCLBV202-GFP monoclonal detection;B:pCLBV202-CB单克隆检测 pCLBV202-CB monoclonal detection。M:2000 bp标准分子量 2000 bp molecular marker;1—10:pCLBV202-GFP转化大肠杆菌单克隆 pCLBV202-GFP transformed E. coli clones;12—18:pCLBV202-CB转化大肠杆菌单克隆 pCLBV202-CB transformed E. coli clones;11、19:水对照 Water control"
图4
pCLBV202-CB对烟草青枯病的防治效果 A:烟草青枯病症状的整体图Overall picture of tobacco bacterial wilt symptoms;B:烟草青枯病分级Classification of tobacco bacterial wilt;C:pCLBV202-CB组烟草青枯病症状Tobacco bacterial wilt symptoms in pCLBV202-CB group;D:pCLBV202组烟草青枯病症状Tobacco bacterial wilt symptoms in pCLBV202 group;E:pCLBV202-CB组注射叶Injected leaf of pCLBV202-CB group;F:pCLBV202组注射叶Injected leaf of pCLBV202 group"
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Cited |
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Shared | ||||||||||||||||||||||||||||||||||||||||||||||||||
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