Cloning and Function Analysis of Mycorrhizal Signaling Receptor Protein Lysin Motif Receptor-like Kinases 2 Gene（LYK2）in Citrus

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

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (2): 281-292.doi: 10.16420/j.issn.0513-353x.2021-0045

• Research Papers • Previous Articles Next Articles

Cloning and Function Analysis of Mycorrhizal Signaling Receptor Protein Lysin Motif Receptor-like Kinases 2 Gene（LYK2）in Citrus

SONG Fang, LI Zixuan, WANG Ce, WANG Zhijing, HE Ligang, JIANG Yingchun, WU Liming(), BAI Fuxi()

Fruit and Tea Subcenter of Hubei Innovation Center of Agricultural Science and Technology,Institute of Fruit and Tea,Hubei Academy of Agricultural Sciences,Wuhan 430064,China

Received:2021-11-09 Revised:2022-01-07 Online:2022-02-25 Published:2022-02-28
Contact: WU Liming,BAI Fuxi E-mail:wuliming2005@126.com;baifx@webmail.hzau.edu.cn

Abstract

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

CLC Number:

S666

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.

Figures/Tables 9

References 44

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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

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

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

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

Cloning and Function Analysis of Mycorrhizal Signaling Receptor Protein Lysin Motif Receptor-like Kinases 2 Gene（LYK2）in Citrus

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