园艺学报 ›› 2023, Vol. 50 ›› Issue (2): 319-330.doi: 10.16420/j.issn.0513-353x.2021-1274
韩睿1,2, 钟雄辉2, 陈登辉1,2, 崔建2, 乐祥庆1,2, 颉建明1,*(), 康俊根2,*(
)
收稿日期:
2022-09-16
修回日期:
2022-11-01
出版日期:
2023-02-25
发布日期:
2023-03-06
通讯作者:
*(E-mail:xiejianminggs@126.com,kangjungen@nercv.org)
基金资助:
HAN Rui1,2, ZHONG Xionghui2, CHEN Denghui1,2, CUI Jian2, YUE Xiangqing1,2, XIE Jianming1,*(), KANG Jungen2,*(
)
Received:
2022-09-16
Revised:
2022-11-01
Online:
2023-02-25
Published:
2023-03-06
Contact:
*(E-mail:xiejianminggs@126.com,kangjungen@nercv.org)
摘要:
野油菜黄单胞菌(Xanthomonas campestris pv. campestris,Xcc)是十字花科作物黑腐病的致病菌。利用酵母双杂交系统,筛选到了野油菜黄单胞菌的效应因子XopR在甘蓝上的靶标蛋白BobHLH34,该蛋白基因的开放阅读框为996 bp,编码331个氨基酸,含有1个保守性较高的碱性/螺旋—环—螺旋结构域(basic/helix1-loop-helix2)。系统进化树分析表明,甘蓝BobHLH34与花椰菜(BrcbHLH34)相似性最高,亚细胞定位结果显示其定位在细胞核内。将野油菜黄单胞菌接种甘蓝叶片,实时荧光定量结果显示BobHLH34的表达量呈现先上升后下降的趋势,接种后16 h表达量达到峰值,为对照的7.7倍;利用甘蓝卷叶病毒载体BobHLH34-PCVA/PCVB真空侵染甘蓝实生苗能够有效降低BobHLH34的表达,导致基因沉默的甘蓝植株对野油菜黄单胞菌株的抗病性比对照显著降低。综上所述,BobHLH34能够正向调控甘蓝对黑腐病的免疫反应。
中图分类号:
韩睿, 钟雄辉, 陈登辉, 崔建, 乐祥庆, 颉建明, 康俊根. 黑腐病菌效应因子XopR的甘蓝靶标基因BobHLH34的克隆及功能分析[J]. 园艺学报, 2023, 50(2): 319-330.
HAN Rui, ZHONG Xionghui, CHEN Denghui, CUI Jian, YUE Xiangqing, XIE Jianming, KANG Jungen. Cloning and Functional Analysis of BobHLH34 Gene in Cabbage that Interacts with XopR from Xanthomonas[J]. Acta Horticulturae Sinica, 2023, 50(2): 319-330.
引物名称 Primer name | 序列(5′-3′) Sequence |
---|---|
XopR-AD-F | GCCATG GAGGCCAGTGAATTCATGTATCAATCAAACGAAGA |
XopR-AD-R | CAGCTCGAGCTCGATGGATCCAGCAGCAAAAGAGCAGTTTTG |
GAPDH-F | CAGGTTTGGAATTGTCGAGG |
GAPDH-R | GAGCTGTGGAAGCACCTTTC |
CE-bhlh34-F | TGGCGCGCCACTAGTGGATCCATGTATCAATCAAACGAAGA |
CE-bhlh34-R-no-TGA | CCCGGGAGCGGTACCCTCGAGTCAAGCAGCAAAAGAGCAGT |
BobHLH34-qRT-F2 | GCTCAAGAAAGAGGGGACGA |
BobHLH34-qRT-R2 | GTTTACGACCCGGATTGCAT |
BobHLH34-PVCA-F | GCCTAAGCGGCTAGCGGTACCCTGCCCTTTGCTTCGATCAA |
BobHLH34-PVCA-R | TAGGCTAGCGAGCTCAGATCTAAACTTGCTCCTGTTCCGTC |
表1 本研究中所用的引物信息
Table 1 Primers used in this study
引物名称 Primer name | 序列(5′-3′) Sequence |
---|---|
XopR-AD-F | GCCATG GAGGCCAGTGAATTCATGTATCAATCAAACGAAGA |
XopR-AD-R | CAGCTCGAGCTCGATGGATCCAGCAGCAAAAGAGCAGTTTTG |
GAPDH-F | CAGGTTTGGAATTGTCGAGG |
GAPDH-R | GAGCTGTGGAAGCACCTTTC |
CE-bhlh34-F | TGGCGCGCCACTAGTGGATCCATGTATCAATCAAACGAAGA |
CE-bhlh34-R-no-TGA | CCCGGGAGCGGTACCCTCGAGTCAAGCAGCAAAAGAGCAGT |
BobHLH34-qRT-F2 | GCTCAAGAAAGAGGGGACGA |
BobHLH34-qRT-R2 | GTTTACGACCCGGATTGCAT |
BobHLH34-PVCA-F | GCCTAAGCGGCTAGCGGTACCCTGCCCTTTGCTTCGATCAA |
BobHLH34-PVCA-R | TAGGCTAGCGAGCTCAGATCTAAACTTGCTCCTGTTCCGTC |
图4 甘蓝BobHLH34在烟草叶片的瞬时表达 A ~ C:BobHLH34-PYBA1132重组表达载体在细胞核中表达;D ~ F:PYBA1132体在细胞核、细胞膜和细胞质中显示绿色荧光。
Fig. 4 The transient expression of BobHLH34 gene in Nicotiana benthamiana leaf epidermal cells A-C:BobHLH34-PYBA1132 recombinant expression vector showed green fluorescence in the nucleus;D-F:PYBA1132 showed green fluorescence in the whole cells.
图8 甘蓝BobHLH34沉默植株对黑腐病的敏感性 A、B:利用携带对照(PCVA/PCVB)和病毒沉默载体(BobHLH34-PCVA/PCVB)农杆菌真空侵染具有两片子叶的甘蓝实生苗,3周后比较发病表型差异;C:利用携带(PDS-PCVA/PCVB)病毒载体的农杆菌真空侵染甘蓝实生苗,作为基因沉默的时间指示标记;D:利用实时荧光定量RT-PCR技术检测BobHLH34的沉默效率;E:Xcc接种8 ~ 12 d后病斑面积测量。* P ≤ 0.05,**P ≤ 0.01.
Fig. 8 Sensitivity analysis of BobHLH34 silenced cabbage plants to Xcc A,B:Cabbage seedlings were infected with Agrobacterium containing empty vectors(PCVA/PCVB)or silenced vectors(BobHLH34-PCVA/PCVB). Leaves were sampled on the three weeks after vacuum infiltration for detection of susceptiblity to Xcc. C:Cabbage seedlings were infected with Agrobacterium containing silenced vectors(PDS-PCVA/PCVB),and the photobleached phenotype was observed in cabbage plants. D:Analysis of BobHLH34 expression by qRT-PCR in silenced plants. E:The lesion area was measured 8-12 days after Xcc inoculation.* P ≤ 0.05,**P ≤ 0.01.
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