黄龙病菌胁迫下‘锦橙’CsCalS5表达和胼胝质沉积的初步分析

doi:10.16420/j.issn.0513-353x.2020-0282

摘要/Abstract

摘要：

胼胝质合成酶是控制胼胝质合成的关键酶,在植物生长发育和抗逆胁迫中具有重要作用。克隆并分析‘锦橙’胼胝质合成酶基因CsCalS5及其启动子序列,实时荧光定量PCR检测CsCalS5的组织表达模式以及植物生长调节剂、黄龙病菌和柑橘溃疡病菌的诱导表达模式,并通过组织切片观察感染黄龙病和柑橘溃疡病‘锦橙’叶脉胼胝质的沉积。结果显示,CsCalS5启动子序列中含有脱落酸和病原菌的响应元件;CsCalS5编码1 952个氨基酸含有保守结构域FKS1和β-1,3-葡聚糖合成酶功能域的跨膜蛋白;CsCalS5在‘锦橙’的茎中高表达,且受脱落酸的诱导;黄龙病菌侵染后叶脉韧皮部的胼胝质沉积明显,患病叶片CsCalS5的表达量是健康对照的4.02倍;柑橘溃疡病菌侵染叶片后,叶脉韧皮部胼胝质沉积无明显增加,且CsCalS5的表达量与健康对照无显著差异。综上,黄龙病菌可能通过上调‘锦橙’CsCalS5的表达促进韧皮部胼胝质的沉积,从而增强其防御能力,且这种抗性可能受到脱落酸的调控。

关键词: 柑橘, 胼胝质合成酶, CsCalS5, 表达分析, 黄龙病

Abstract:

Callose synthase is a key enzyme to control callose synthesis and plays an important role in plant growth and stress resistance. The CsCalS5 gene and promoter sequence of Citrus sinensis‘Jincheng’ were cloned and analyzed. Its tissue expression and induced expression with exogenous phytohormones,Candidatus Liberibacter asiaticus（CLas）and Xanthomonas citri subsp. Citri（Xcc）were detected by real-time fluorescent quantitative PCR. The callose deposition in vein of CLas-infected or Xcc-infected C. sinensis was observed by tissue section staining. The CsCalS5 promoter sequence contained abscisic acid （ABA）and pathogen response elements. The CsCalS5 gene encoded a transmembrane protein composed of 1 952 amino acids containing the conserved domains FKS1 and β-1,3-glucan synthetase functional domain. CsCalS5 was highly expressed in the stem of C. sinensis,and could be induced by ABA. The expression of CsCalS5 in CLas-infected leaves was 4.02 times of that in the healthy control,and the callose was obviously deposited in the phloem of vein of CLas-infected leaves. After Xcc infection,callose deposition was not significantly increased in the veins phloem,the expression of CsCalS5 was not significantly different from healthy controls. In conclusion,CLas might promote the deposition of callose in the phloem by up-regulating the expression of CsCalS5 in C. sinensis,which could be regulated by ABA.

Key words: Citrus sinensis, callose synthase, CsCalS5, expression analysis, Huanglongbing

中图分类号:

S666

张庆雯, 祁静静, 谢宇, 谢竹, 彭蕴, 李强, 彭爱红, 邹修平, 何永睿, 陈善春, 姚利晓. 黄龙病菌胁迫下‘锦橙’CsCalS5表达和胼胝质沉积的初步分析[J]. 园艺学报, 2021, 48(2): 276-288.

ZHANG Qingwen, QI Jingjing, XIE Yu, XIE Zhu, PENG Yun, LI Qiang, PENG Aihong, ZOU Xiuping, HE Yongrui, CHEN Shanchun, YAO Lixiao. Preliminary Analysis of CsCalS5 and Callose Deposition in Citrus sinensis Infected with Candidatus Liberibacter asiaticus[J]. Acta Horticulturae Sinica, 2021, 48(2): 276-288.

图/表 10

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引物名称 Primer name	引物序列（5′-3′） Primer sequence
CsCalS5-1	F：ATAGAGGCAAAAGATGTCACAGAGA;R：TTTTGCCGATCCATCTGTCAC
CsCalS5-2	F：CGTGCTGACGCTGATTTCT;R：AATAACCTCTTTCTCTCGTTCTCC
CsCalS5-3	F：GCTAGCAAGATACCCATAGCATT;R：CTTAAGGAATTCTTGAAGCAAATTC
CsCalS5-4	F：GGAAACCTGAAAATCAAAACCA;R：TCACTCCTTACTTTTGGAAGACCT
CsCalS5P	F：CTCATGCTTTATGCCTTT;R：CTTTTGCCTCTATAAATATGAT
Actin	F：CATCCCTCAGCACCTTCC;R：CCAACCTTAGCACTTCTCC
qCsCalS5	F：AACGTTCTGGTGATGCTCGT;R：AGCCTTCGCAAGAGCTGAAT
OI1/OI2C	F：GCGCGTATGCAATACGAGCGGCA;R：GCCTCGCGACTTCGCAACCCAT

引物名称 Primer name	引物序列（5′-3′） Primer sequence
CsCalS5-1	F：ATAGAGGCAAAAGATGTCACAGAGA;R：TTTTGCCGATCCATCTGTCAC
CsCalS5-2	F：CGTGCTGACGCTGATTTCT;R：AATAACCTCTTTCTCTCGTTCTCC
CsCalS5-3	F：GCTAGCAAGATACCCATAGCATT;R：CTTAAGGAATTCTTGAAGCAAATTC
CsCalS5-4	F：GGAAACCTGAAAATCAAAACCA;R：TCACTCCTTACTTTTGGAAGACCT
CsCalS5P	F：CTCATGCTTTATGCCTTT;R：CTTTTGCCTCTATAAATATGAT
Actin	F：CATCCCTCAGCACCTTCC;R：CCAACCTTAGCACTTCTCC
qCsCalS5	F：AACGTTCTGGTGATGCTCGT;R：AGCCTTCGCAAGAGCTGAAT
OI1/OI2C	F：GCGCGTATGCAATACGAGCGGCA;R：GCCTCGCGACTTCGCAACCCAT

元件名称 Element name	序列 Sequence	数量 Number	功能 Function	位置 Location
GT1 box	GAAAAA	2	病原菌和盐响应 Pathogen or salt-induced responsiveness	-264（+）/-976（-）
dof box	AAAG	23	病原菌响应Pathogen responsiveness	-1281/-1178/-1097/-974/-927/ -838/-260/-161/-148/-81/-7（+）-1486/-1340/-1264/-1060/-869/-707/-656/-610/-545/-479/-207/-85（-）
W box	TGACT	2	病原菌和损伤响应 Pathogen and wound responsiveness	-474（+）/-582（+）
GARE-motif	TCTGTTG	2	赤霉素响应元件Gibberellin-responsive element	-281（+）
	CCTTTT	2	赤霉素响应元件Gibberellin-responsive element	-1490（+）
TCA-element	CCATCTTTTT	1	水杨酸响应元件Salicylic acid responsiveness	-1067（+）
ABRE	ACGTG	1	脱落酸响应元件Abscisic acid responsiveness	-814（+）
WUN-motif	AAATTACTA	1	损伤响应元件Wound responsiveness	-1369（-）
MYC	CATTTG	2	干旱和冻害响应元件 Drought and freeze responsiveness	-1473（+）/-1400（-）
MBS	CAACTG	1	参与干旱诱导Involved in drought-inducibility	-1259（-）
ARE	AAACCA	2	厌氧诱导所需Essential for the anaerobic induction	-1255（+）/-456（-）
MBSI	TTTTTACGGTTA	1	参与类黄酮合成基因的调控 Involved in flavonoid biosynthetic genes regulation	-131（+）
RY-element	CATGCATG	1	参与种子特异性调控 Involved in seed-specific regulation	-696（-）
GATA-motif	AAGATAAGATT	1	光响应元件Light responsiveness	-1400（-）
GT1-motif	GGTTAA	1	光响应元件Light responsiveness	-1336（-）
chs-CMA2a	TCACTTGA	1	光响应元件Light responsiveness	-1268（-）
Box 4	ATTAAT	1	光响应元件Light responsiveness	-1293（+）
LAMP-element	CTTTATCA	1	光响应元件Light responsiveness	-1123（+）
G-box	TACGTG	2	光响应元件Light responsiveness	-810（+）
G-box	GGTTAAT	2	光响应元件Light responsiveness	-194（+）

元件名称 Element name	序列 Sequence	数量 Number	功能 Function	位置 Location
GT1 box	GAAAAA	2	病原菌和盐响应 Pathogen or salt-induced responsiveness	-264（+）/-976（-）
dof box	AAAG	23	病原菌响应Pathogen responsiveness	-1281/-1178/-1097/-974/-927/ -838/-260/-161/-148/-81/-7（+）-1486/-1340/-1264/-1060/-869/-707/-656/-610/-545/-479/-207/-85（-）
W box	TGACT	2	病原菌和损伤响应 Pathogen and wound responsiveness	-474（+）/-582（+）
GARE-motif	TCTGTTG	2	赤霉素响应元件Gibberellin-responsive element	-281（+）
	CCTTTT	2	赤霉素响应元件Gibberellin-responsive element	-1490（+）
TCA-element	CCATCTTTTT	1	水杨酸响应元件Salicylic acid responsiveness	-1067（+）
ABRE	ACGTG	1	脱落酸响应元件Abscisic acid responsiveness	-814（+）
WUN-motif	AAATTACTA	1	损伤响应元件Wound responsiveness	-1369（-）
MYC	CATTTG	2	干旱和冻害响应元件 Drought and freeze responsiveness	-1473（+）/-1400（-）
MBS	CAACTG	1	参与干旱诱导Involved in drought-inducibility	-1259（-）
ARE	AAACCA	2	厌氧诱导所需Essential for the anaerobic induction	-1255（+）/-456（-）
MBSI	TTTTTACGGTTA	1	参与类黄酮合成基因的调控 Involved in flavonoid biosynthetic genes regulation	-131（+）
RY-element	CATGCATG	1	参与种子特异性调控 Involved in seed-specific regulation	-696（-）
GATA-motif	AAGATAAGATT	1	光响应元件Light responsiveness	-1400（-）
GT1-motif	GGTTAA	1	光响应元件Light responsiveness	-1336（-）
chs-CMA2a	TCACTTGA	1	光响应元件Light responsiveness	-1268（-）
Box 4	ATTAAT	1	光响应元件Light responsiveness	-1293（+）
LAMP-element	CTTTATCA	1	光响应元件Light responsiveness	-1123（+）
G-box	TACGTG	2	光响应元件Light responsiveness	-810（+）
G-box	GGTTAAT	2	光响应元件Light responsiveness	-194（+）