金柑UDP-鼠李糖合成酶基因的克隆与功能解析

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

摘要/Abstract

摘要：

以‘浏阳金柑’为研究材料,克隆到2个UDP-鼠李糖合成酶（UDP-Rhamnose synthase,RHM）基因FcRHM1和FcRHM2的全长。FcRHM1的开放阅读框（ORF）长度为2 007 bp,编码668个氨基酸,分子量为75.3 kD;FcRHM2的ORF长度为2 031 bp,编码676个氨基酸,分子量为76.2 kD。多序列比对显示,FcRHM与其他植物的RHM蛋白序列高度相似。亚细胞定位显示FcRHM1和FcRHM2定位于细胞质和细胞核。原核表达及体外酶促反应结果显示,FcRHM1和FcRHM2均可将UDP-葡萄糖转化为UDP-鼠李糖。组织特异性表达显示,FcRHM1和FcRHM2在不同组织中表达量存在一定的差异。结合UDP-鼠李糖的积累模式,认为FcRHM1和FcRHM2可能都参与了UDP-鼠李糖的合成,但FcRHM1对于花和果实中UDP-鼠李糖的合成更为关键,而FcRHM2则主要负责叶片及芽的UDP-鼠李糖的合成。

关键词: 金柑, UDP-鼠李糖, UDP-鼠李糖合成酶, 基因功能

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

中图分类号:

S666

杨禹妍, 段新圆, 何治霖, 邴起浩, 陈锁英, 刘晓曼, 曾明, 刘小刚. 金柑UDP-鼠李糖合成酶基因的克隆与功能解析[J]. 园艺学报, 2022, 49(8): 1663-1672.

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.

图/表 7

表1 引物序列

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

图1 金柑FcRHM与其他植物RHM同源蛋白的序列比对 Fc：金柑;At：拟南芥;Pp：杨树;Vv：葡萄。

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

图2 金柑FcRHM1和FcRHM2蛋白的亚细胞定位

Fig. 2 Subcellular localization of FcRHM1 and FcRHM2 proteins

图3 金柑FcRHM1和FcRHM2 蛋白SDS-PAGE电泳图 1：沉淀中FcRHM1;2：上清液中FcRHM1;3：沉淀中FcRHM2;4：上清液中FcRHM2。箭头所指为重组蛋白。

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.

图4 FcRHM-N和FcRHM-C纯化蛋白SDS-PAGE电泳图 1：FcRHM1-N蛋白;2：FcRHM1-C蛋白;3：FcRHM2-N蛋白;4：FcRHM2-C蛋白。

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

图5 HPLC检测FcRHM-N和FcRHM-C催化产物

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

图6 FcRHM1和FcRHM2表达量和UDP-鼠李糖含量

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

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