柑橘菌根信号受体蛋白基因LYK2的克隆及功能分析

doi:10.16420/j.issn.0513-353x.2021-0045

摘要/Abstract

摘要：

从甜橙基因组中鉴定到12个菌根信号受体蛋白基因CsLYK,分布于chr1、chr2、chr6、chr7、chr8和chr9等6条染色体上。结构域分析表明CsLYK家族基因的3′端含有7个保守motif,且其组合也高度保守;而5′端仅有3个保守motif,且其组合变异较大。另外,CsLYK2、CsLYK3、CsLYK6、CsLYK10和CsLYK12没有内含子,其余7个成员含有1 ~ 19个内含子。系统进化分析将CsLYK基因分为3组,CsLYK2与已经报道的菌根共生信号受体基因PaNFP和SlLYK10处于同一小分支。qRT-PCR分析表明,枳实生苗中12个PtrLYK在根、茎、叶中均有表达,其中PtrLYK10在根系中高量表达,PtrLYK3、PtrLYK7、PtrLYK8、PtrLYK9和PtrLYK12在叶片中高量表达;另外,PtrLYK2在枳根系中的表达受菌根侵染的诱导,且在菌根侵染的早期表达量最高。在拟南芥原生质体中瞬时表达亚细胞定位载体35S-PtrLYK2-CFP,共聚焦荧光显微镜观察发现PtrLYK2定位于细胞膜上。通过苜蓿毛状根瞬时表达启动子表达载体proPtrLYK2::GUS,利用Ds-Red红色荧光报告基因筛选阳性根并接种AM真菌,通过GUS组织化学染色和WGA-488菌根染色发现proPtrLYK2主要在丛枝周围的细胞和根尖细胞表达。这些结果表明PtrLYK2可能在柑橘菌根共生早期的信号识别中发挥重要功能。

关键词: 柑橘, 丛枝菌根真菌, LysM受体类似蛋白激酶, 亚细胞定位, 启动子

Abstract:

Twelve CsLYK genes were annotated from Citrus sinensis genome,which were unevenly distributed on six chromosomes,including chr1,chr2,chr6,chr7,chr8 and chr9. The conserved domain analysis showed that seven conserved motifs were detected at 3′ terminator of CsLYKs,and the constructions of these seven motifs were highly conserved. However,only three conserved motifs were detected at 5′ terminator of CsLYKs,and the constructions of these three motifs were highly variable. Gene structure analysis showed that CsLYK2,CsLYK3,CsLYK6,CsLYK10 and CsLYK12 had no introns,while other seven CsLYK family members had one to nineteen introns. Phylogenetic analysis revealed that twelve CsLYKs could be divided into three groups and CsLYK2 was clustered together with PaNFP and SlLYK10 in a small brunch. The expression analysis revealed that all PtrLYK genes were expressed in the root,stem and leaf of P. trifoliata. In details,the expression of PtrLYK10 was more abundant in the roots than leaf and stem,and the expression of PtrLYK3,PtrLYK7,PtrLYK8,PtrLYK9 and PtrLYK12 were more abundant in the leaf. Additionally,the expression of PtrLYK2 was induced in the roots of poncirus during different mycorrhizal infection periods,and the highest expression level was observed at two weeks post inoculation. The subcellular localization vector,35S-PtrLYK2-CFP,was transiently expressed in the protoplast of Arabidopsis thaliana. The result of laser scanning Confocal microscope indicated that PtrLYK2 was expressed on the plasma membrane. The promoter expression vector,proPtrLYK2::GUS, was transiently expressed in M. truncatula hair root,and the positive roots were screened with Ds-Red report gene and inoculated with AM fungi. After GUS histochemical staining and WGA-488 mycorrhizal staining,the expression of proPtrLYK2 was observed in the cells around arbuscules and the root tip cells. Taken together,these results indicated that PtrLYK2 played a significant role in symbiotic signaling recognition at early stage between citrus and AM fungi.

Key words: Citrus, arbuscular mycorrhizal fungi, LysM receptor-like kinases, subcellular localization, promoter

中图分类号:

S666

宋放, 李子璇, 王策, 王志静, 何利刚, 蒋迎春, 吴黎明, 白福玺. 柑橘菌根信号受体蛋白基因LYK2的克隆及功能分析[J]. 园艺学报, 2022, 49(2): 281-292.

SONG Fang, LI Zixuan, WANG Ce, WANG Zhijing, HE Ligang, JIANG Yingchun, WU Liming, BAI Fuxi. Cloning and Function Analysis of Mycorrhizal Signaling Receptor Protein Lysin Motif Receptor-like Kinases 2 Gene（LYK2）in Citrus[J]. Acta Horticulturae Sinica, 2022, 49(2): 281-292.

图/表 9

表1 实时荧光定量PCR引物

Table 1 The primers used for qRT-PCR

登录号 Accession number	基因 Gene	正向引物序列（5′-3′） Forward primer sequence	反向引物序列（5′-3′） Reverse primer sequence
Cs1g15820.1	PtrLYK1	GCAGATTGCACTTGATGCTG	CTTTGCTCGAAAAGCACTGTC
Cs1g23560.1	PtrLYK2	CCACAATTGATGGGAAGGTC	ACTTCACCAGATTGGCATGG
Cs1g23580.1	PtrLYK3	TGTTGGACTTGCCACAACAG	ACGGTGTTGAAAGGAGGTTG
Cs2g02680.1	PtrLYK4	ATCCGATTGAGCTTGGTGAG	GGATGAAAACCAAGCCACTG
Cs2g20910.1	PtrLYK5	AGTCGATTGCGAGGAATGTG	CCCGGAATAAAGACCAAACC
Cs2g25010.1	PtrLYK6	GAACCCCAGTTATGTTCACAGG	AATTGTGGGCTCTCGTTGTC
Cs6g02700.1	PtrLYK7	TTTGAGTGTGGTGGTTGTGG	TGTTGTTGCAGCAAGTAGCC
Cs6g02710.1	PtrLYK8	CCATGGATCTTGAAGGGTTG	TCCTCTGGCACGGAAAATAC
Cs7g09310.1	PtrLYK9	TCACACGAAAGCTCGATACG	TTCCTCAAGCTTCACCAACC
Cs8g16260.1	PtrLYK10	CCCGATGATTTCTCAAGCAG	GGAGATCCCGAATTTCATCC
Cs9g08050.1	PtrLYK11	AATCCCAGTGCCTTTCCTTC	TTTGGCATGGATAGGTGGTC
orange1.1t04036.1	PtrLYK12	TGAAGTGGTGTTGGTGTTGG	CGATGGATACAATCCGTTGC
Cs1g05000.1	β-actin	AGAACTATGAACTGCCTGATGGC	GCTTGGAGCAAGTGCTGTGATT

表2 PtrLYK2基因CDS和启动子扩增引物

Table 2 The primers used for cloning CDS and promoter sequence of PtrLYK2

用途 Purpose	引物名称 Primer name	引物序列（5′-3′） Primer sequence
PtrLYK2 CDS序列扩增 Amplification of coding sequence of PtrLYK2	PtrLYK2-F	ATGGCAATTTCTTCTCTTTCCT
	PtrLYK2-R	GCGAGCTGTGACTGGACTAA
PtrLYK2启动子序列扩增 Amplification of promoter sequence of PtrLYK2	proPtrLYK2-F	GATAGAACACTTTGCGGTC
	proPtrLYK2-R	TGGTACAAATGTTCTTTTGT

图1 甜橙LYK家族基因的染色体定位

Fig. 1 The localization of LYK family genes in citrus genome

图2 甜橙LYK家族基因的保守结构域（A）和基因结构（B）

Fig. 2 The conserved motifs（A）and gene structure（B）of LYK family genes in citrus

图3 LYK基因家族的系统进化树 Cs：柑橘;Sl：番茄;At：拟南芥;Pa：糙叶山黄麻;Gm：大豆;Lj：百脉根;Os：水稻;Pt：杨树;Vv：葡萄;Mt：苜蓿。

Fig. 3 The phylogenetic tree of LYK family genes Cs：Citrus sinensis;Sl：Solanum lycopersicum;At：Arabidopsis thaliana;Pa： Parasponia andersonii;Gm：Glycine max;Lj：Lotus japonicus;Os：Oryza sativa;Pt：Populus trichocarpa;Vv：Vitis vinifera;Mt：Medicago truncatula.

图4 PtrLYK在枳实生苗根、茎、叶中的表达量

Fig. 4 The expression of PtrLYK family genes in the root,stem and leaf of Poncirus trifoliata seedlings

图5 菌根真菌侵染不同时期枳根系PtrLYK2的表达分析

Fig. 5 The expression analysis of PtrLYK2 during different mycorrhizal infection periods in the roots of Poncirus trifoliate

图6 PtrLYK2的拟南芥原生质体中的亚细胞定位

Fig. 6 The subcellular localization of PtrLYK2 in protoplast of Arabidopsis thaliana

图7 PtrLYK2启动子的GUS组织化学染色（A、B）和丛枝菌根真菌的WGA-488染色（C） A：proPtrLYK2:GUS在根尖细胞表达;B：proPtrLYK2:GUS在丛枝细胞及周围细胞表达;C：丛枝菌根真菌的丛枝细胞（Ar）WGA-488染色。

Fig. 7 The GUS histochemical staining of the PtrLYK2 promoter（A,B）and WGA-488 staining of arbuscular mycorrhizal fungi（C） A：proPtrLYK2:GUS was expressed in the root tip cells;B：proPtrLYK2:GUS was expressed in the arbuscules and cells around arbuscules;C：The WGA-488 staining of arbuscules（Ar）of arbuscular mycorrhizal fungi.

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