Cloning and Function Characterization of UDP-L-rhamnose Synthase from Fortunella crassifolia

doi:10.16420/j.issn.0513-353x.2021-0560

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (8): 1663-1672.doi: 10.16420/j.issn.0513-353x.2021-0560

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Cloning and Function Characterization of UDP-L-rhamnose Synthase from Fortunella crassifolia

YANG Yuyan¹^,², DUAN Xinyuan¹^,², HE Zhilin¹, BING Qihao¹, CHEN Suoying¹, LIU Xiaoman¹, ZENG Ming¹^,², LIU Xiaogang¹^,²^,^*()

1. College of Horticulture and Landscape Architecture,Southwest University,Chongqing 400715,China
2. Key Laboratory of Horticulture Science for Southern Mountainous Regions,Ministry of Education,Chongqing 400715,China

Received:2022-02-23 Revised:2022-06-06 Online:2022-08-25 Published:2022-09-05
Contact: LIU Xiaogang E-mail:lxgz16853@163.com

Abstract

Abstract:

Two UDP-rhamnose synthase（RHM）genes（FcRHM1 and FcRHM2）were cloned from Fortunella crassifolia. The open reading frame（ORF）of FcRHM1 gene was 2 007 bp,encoding protein consisting of 668 amino acids with 75.3 kD,and FcRHM2 was 2 031 bp encoding 676 amino acids with 76.2 kD. In vitro experiments showed that FcRHM1 and FcRHM2 could function in converting UDP-glucose into UDP-rhamnose（UDP-Rha）. Tissue-specific expressions revealed that the relative expression levels of FcRHM1 and FcRHM2 are different in different tissues. Combination with accumulation patterns of UDP-Rha,we concluded that both FcRHM1 and FcRHM2 were involved in the biosynthesis of UDP-Rha. Among them,FcRHM1 might be more critical in UDP-Rha of flowers and fruits,whereas FcRHM2 is mainly responsible for leaves and buds.

Key words: Fortunella crassifolia, UDP-rhamnose, UDP-rhamnose synthase, gene function

CLC Number:

S666

YANG Yuyan, DUAN Xinyuan, HE Zhilin, BING Qihao, CHEN Suoying, LIU Xiaoman, ZENG Ming, LIU Xiaogang. Cloning and Function Characterization of UDP-L-rhamnose Synthase from Fortunella crassifolia[J]. Acta Horticulturae Sinica, 2022, 49(8): 1663-1672.

Figures/Tables 7

Table 1 Oligonucleotide sequences for primers

引物用途 Purpose	基因 Gene	正向引物（5′-3′） Forward primer sequence	反向引物（5′-3′） Reverse primer sequence
ORF扩增 ORF amplification	FcRHM1	ATGGCCACGTATACCCCGAAGAAC	CTAGGTTTTCTTGTTGGGCTCAAAG
	FcRHM2	ATGGCTACTGTTTACACGCCCAAG	CTATTTTGCCACTCCACCGAAAGTGG
qRT-PCR	FcRHM1	CCTCCTTGAAGCCTGCAAAGTG	CAGGGAACTGATTTGGTCCATAAAC
	FcRHM2	GCAAAAGACAATGAAATGGTAC	TTGAGCATGAAAGTAGTATCATG
	β-Actin	ATCTGCTGGAAGGTGCTGAG	CCAAGCAGCATGAAGATCAA

Fig. 1 Alignment of FcRHM with other plants RHM proteins Fc：Fortunella crassifolia;At：Arabidopsis thaliana;Pp：Prunus persica;Vv：Vitis vinifera.

Fig. 2 Subcellular localization of FcRHM1 and FcRHM2 proteins

Fig. 3 SDS-PAGE of FcRHM1 and FcRHM2 proteins 1：FcRHM1 from precipitate;2：FcRHM1 from supernatant;3：FcRHM2 from precipitate;4：FcRHM2 from supernatant. Arrow represents recombinant proteins.

Fig. 4 SDS-PAGE of FcRHM-N and FcRHM-C proteins 1：FcRHM1-N;2：FcRHM1-C;3：FcRHM2-N：4：FcRHM2-C.

Fig. 5 HPLC analyses of the reaction products of FcRHM-N and FcRHM-C

Fig. 6 Expression of FcRHM1 and FcRHM2 and accumulation of UDP-Rha

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Cloning and Function Characterization of UDP-L-rhamnose Synthase from Fortunella crassifolia

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