中国农业科学 ›› 2019, Vol. 52 ›› Issue (4): 639-650.doi: 10.3864/j.issn.0578-1752.2019.04.006
胡安华,祁静静,张庆雯,陈善春,邹修平,许兰珍,彭爱红,雷天刚,姚利晓,龙琴,何永睿(),李强(
)
收稿日期:
2018-10-13
接受日期:
2018-11-26
出版日期:
2019-02-16
发布日期:
2019-02-27
通讯作者:
何永睿,李强
作者简介:
胡安华,E-mail: 基金资助:
HU AnHua,QI JingJing,ZHANG QingWen,CHEN ShanChun,ZOU XiuPing,XU LanZhen,PENG AiHong,LEI TianGang,YAO LiXiao,LONG Qin,HE YongRui(),LI Qiang(
)
Received:
2018-10-13
Accepted:
2018-11-26
Online:
2019-02-16
Published:
2019-02-27
Contact:
YongRui HE,Qiang LI
摘要:
【目的】克隆CsPGIP并分析其表达特性,转化柑橘得到超表达转基因株系,并进行柑橘溃疡病抗性评价,为柑橘溃疡病分子育种提供理论依据。【方法】从晚锦橙和四季橘中克隆柑橘CsPGIP,使用MEGA6进行多序列比对并构建系统发育树;采用在线软件BaCelLo和SignalP 4.0进行亚细胞定位和信号肽预测并用GFP瞬时表达确定CsPGIP在细胞内的定位;利用实时荧光定量PCR(qRT-PCR)比较接种溃疡病菌前后高感品种和高抗品种中柑橘CsPGIP的表达特性,分析溃疡病菌侵染与CsPGIP表达的相关性;农杆菌介导遗传转化晚锦橙,采用GUS染色初筛、PCR鉴定和qRT-PCR相结合的方法鉴定超表达转基因株系;观察转基因和野生型株系表型变化,分析其株高、叶片表型;离体针刺法对超表达转基因株系和野生型株系进行柑橘溃疡病抗性评价,统计病斑面积和病情指数,分析CsPGIP表达对柑橘抗、感溃疡病的影响。【结果】晚锦橙和四季橘CsPGIP均编码328个氨基酸,与已报道的柑橘中的PGIP同源性高达99.39%,都包含2个PGIP基因典型的LRR结构域(LRR_1和LRR_2);构建系统进化树发现甜橙中的CsPGIP与葡萄中的PGIP(GSVIVT01033370001)遗传距离最近,相似度达到62.97%,推测CsPGIP与葡萄中的PGIP具有类似的抗病效果。亚细胞定位和信号肽预测结果表明CsPGIP属于分泌蛋白,GFP洋葱瞬时表达证明柑橘CsPGIP定位在细胞膜和细胞壁,与预测结果一致。高感品种晚锦橙和高抗品种四季橘接种溃疡病菌后CsPGIP的表达特性不同,在高感品种中表达显著下调,而高抗品种中表达显著上调且维持在较高水平,推测CsPGIP与柑橘溃疡病的抗性相关。构建CsPGIP超表达载体并转化晚锦橙,通过PCR鉴定和qRT-PCR确定其中9个(OE1、OE3、OE4、OE5、OE6、OE9、OE10、OE12和OE14)为CsPGIP超表达阳性株系。通过对转基因株系的表型观察发现OE3、OE14株系表型与野生型株系相比差异明显,植株表现为较矮小,其中OE14出现叶片卷曲、增厚的表型变化。对CsPGIP超表达转基因株系(8个株系)进行离体抗溃疡病评价,结果显示超表达转基因株系可以使柑橘溃疡病病斑面积降至野生型的24.11%—83.88%,其中OE1株系的病斑面积最小;从病情指数来看,除OE3株系外,其余株系的病情指数均比野生型显著下降(为野生型的23.12%—75.49%),其中OE1下降最显著,综上结果可知超表达CsPGIP可以有效抑制柑橘溃疡病菌的生长。【结论】CsPGIP是柑橘响应溃疡病菌侵染的重要基因,可抑制或减轻柑橘溃疡病的发病程度,在柑橘抗溃疡病机理研究方面具有较大的应用价值,也可作为柑橘抗溃疡病分子育种的一个候选基因。
胡安华,祁静静,张庆雯,陈善春,邹修平,许兰珍,彭爱红,雷天刚,姚利晓,龙琴,何永睿,李强. 柑橘溃疡病相关基因CsPGIP的克隆与表达[J]. 中国农业科学, 2019, 52(4): 639-650.
HU AnHua,QI JingJing,ZHANG QingWen,CHEN ShanChun,ZOU XiuPing,XU LanZhen,PENG AiHong,LEI TianGang,YAO LiXiao,LONG Qin,HE YongRui,LI Qiang. Cloning and Expression Analysis of the Citrus Bacterial Canker-Related Gene CsPGIP in Citrus[J]. Scientia Agricultura Sinica, 2019, 52(4): 639-650.
表1
本研究使用的引物"
引物名称Primer name | 引物序列Primer sequence (5′-3′) | 酶切位点Enzyme site |
---|---|---|
OE-CsPGIP-f | CGGGATCCATGAGCAACACGTCACTGTTGTCT | BamHI |
OE-CsPGIP-r | CGGAATTCTCACTTGCAGCTTTCGAGGGGCGC | EcoRI |
SCL-CsPGIP-f | GGGGTACCATGAGCAACACGT CACTGTTGT | KpnI |
SCL-CsPGIP-r | TCCCCCGGGCTTGCAGCTTTCG AGGGGCGCG | SmaI |
qPCR-CsPGIP-f | AGAAGCTTGGCGCTCTTCAT | N/A |
qPCR-CsPGIP-r | TCGCCTTCAAGCTTGTTCCT | N/A |
qPCR-Actin-f | CATCCCTCAGCACCTTCC | N/A |
qPCR-Actin-r | CCAACCTTAGCACTTCTCC | N/A |
OE-f (35S) | AGTAAGGATCGAT CCCACAAAGT | N/A |
OE-r (CsPGIP) | TTTTTGAAGAGTAGTGAAGCTGCA | N/A |
图3
CsPGIP的亚细胞定位 A1:明视野观察CsPGIP-GFP融合蛋白Image of CsPGIP-GFP under bright field;A2:暗视野观察CsPGIP-GFP融合蛋白Image of CsPGIP-GFP under dark field;A3:A1、A2视野叠加Overlap of A1 and A2;B1:明视野观察CsPGIP-GFP融合蛋白质壁分离Image of CsPGIP-GFP under bright field (plasmolysis);B2:暗视野观察CsPGIP-GFP融合蛋白质壁分离Image of CsPGIP-GFP under dark field (plasmolysis);B3:B1、B2视野叠加Overlap of B1 and B2;C1:明视野观察GFP表达Image of GFP under bright field;C2:暗视野观察GFP表达Image of GFP under dark field;C3:C1、C2视野叠加Overlap of C1 and C2;D1:明视野观察GFP质壁分离GFP of plasmolysis under bright field;D2:暗视野观察GFP质壁分离GFP of plasmolysis under dark field;D3:D1、D2视野叠加Overlap of D1 and D2;标尺Scale:100 μm"
图5
转基因株系的鉴定及CsPGIP表达分析 A:CsPGIP转基因株系的PCR鉴定PCR amplification of CsPGIP in over-expression transgenic lines;B:转基因株系中CsPGIP的相对表达量检测The relative expression level of CsPGIP in over-expression transgenic lines。 M:分子量标准Marker;OE1—OE14:GUS初筛的转基因材料Lines verified from GUS staining;WT:野生型Wild-type;阳性株系特异扩增条带为1 530 bp PCR product size of positive lines is 1 530 bp"
图6
转基因株系的表型分析 A:树龄1年的转基因株系(OE1、OE3、OE4)和野生型对照(WT1)植株The phenotype of 1-year-old transgenic lines (OE1, OE3, OE4) and the wild-type control (WT1);B:树龄6个月的转基因株系(OE5、OE6、OE9、OE10、OE12、OE14)和野生型对照(WT2)植株Phenotype of 6-month-old transgenic lines (OE5, OE6, OE9, OE10, OE12, OE14) and the wild-type control (WT2);C:野生型对照WT2和转基因株系OE14的叶片Leaves of WT2 and OE14;D:树龄1年的转基因株系(OE1、OE3、OE4)和野生型对照(WT1)的株高(测量方法:从嫁接口到顶梢的距离)Height of 1-year-old transgenic lines (OE1, OE3, OE4) and the wild-type control (measurement method: distance from the graft to the top tip);E:树龄6个月的转基因株系(OE5、OE6、OE9、OE10、OE12、OE14)和野生型对照(WT2)的株高Height of 6-month-old transgenic lines (OE5, OE6, OE9, OE10, OE12, OE14) and the wild-type control"
图7
转基因株系的抗性评价 A:接种LB培养基的转基因株系和野生型对照叶片Disease spots of transgenic lines and the wild-type inoculated with LB;B:接种溃疡病菌的转基因株系和野生型对照叶片Disease spots of transgenic lines and the wild-type inoculated with Xcc;C:接种溃疡病菌的转基因株系和野生型对照病斑面积Lesion area of transgenic lines and the wild-type inoculated with Xcc;D:接种溃疡病菌的转基因株系和野生型对照病情指数Disease index of transgenic lines and the wild-type inoculated with Xcc。WT:野生型对照Wild-type control;OE1—OE12:转基因株系Transgenic lines。*表示差异显著(P<0.05),**表示差异极显著(P<0.01)* represents significant difference (P<0.05), ** represents extremely significant difference (P<0.01)"
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