Screening and Identification of LCD-interacting Proteins in Rice

doi:10.13560/j.cnki.biotech.bull.1985.2020-0510

Abstract

Abstract:

Low cadmium（LCD）gene is involved in grain Cd accumulation of rice. To screen LCD-interacting proteins is beneficial for revealing the molecular mechanism of LCD-mediated grain Cd accumulation and regulation. Using Oryza sativa ssp. japonica cv. Nipponbare as experimental material,the cDNA libraries of yeast two hybrid membrane system and nuclear system were constructed,and LCD was used as bait protein to screen the membrane system and nuclear system respectively. A total of 29 genes were obtained,and the gene ontology（GO）analyses were conducted to annotate their molecular function. Further,these genes were cloned and one-to-one interaction verification with LCD was performed. Finally,a total of 21 LCD-interacting proteins including 16 from membrane system and 5 from nuclear system were identified. Among them,6 proteins were related to the tolerance and transport of metal ions,5 were related to the stress responses,4 were related to plant development responses,and the rest 6 were related to cellular and physiological metabolisms.

Key words: rice, LCD, low cadmium, yeast two-hybrid（Y2H）, interacting protein

HAO Xiao-hua, DAI Jia-li, JI Wen-jin, HUANG Dan, LI Dong-ping, TIAN Lian-fu. Screening and Identification of LCD-interacting Proteins in Rice[J]. Biotechnology Bulletin, 2020, 36(11): 21-29.

Figures/Tables 12

References 44

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引物名称	引物序列（5'-3'）	酶切位点
pBT3-N-F	GGCCATTACGGCCATGGTGGAAACCACAGTT	Sfi I
pBT3-N-R	GGCCGAGGCGGCCTTTTTTACAAGGCCACTT	Sfi I
pGBKT7-F	GGAATTCATGGTGGAAACCACAGTTGATGTT	EcoR I
pGBKT7-R	CGGGATCCGTTTTTTACAAGGCCACTTCCAG	BamH I

引物名称	引物序列（5'-3'）	酶切位点
pBT3-N-F	GGCCATTACGGCCATGGTGGAAACCACAGTT	Sfi I
pBT3-N-R	GGCCGAGGCGGCCTTTTTTACAAGGCCACTT	Sfi I
pGBKT7-F	GGAATTCATGGTGGAAACCACAGTTGATGTT	EcoR I
pGBKT7-R	CGGGATCCGTTTTTTACAAGGCCACTTCCAG	BamH I

引物名称	引物序列（5'-3'）
pPR3-N-F	GTCGAAAATTCAAGACAAG
pPR3-N-R	AAGCGTGACATAACTAATTA
pGADT7-F	GGAGTACCCATACGACGTACC
pGADT7-R	TATCTACGATTCATCTGCAGC

引物名称	引物序列（5'-3'）
pPR3-N-F	GTCGAAAATTCAAGACAAG
pPR3-N-R	AAGCGTGACATAACTAATTA
pGADT7-F	GGAGTACCCATACGACGTACC
pGADT7-R	TATCTACGATTCATCTGCAGC

组合	质粒1	质粒2	目的	缺陷培养基	生长状态
1	pBT3-N-LCD	-	自激活检测	SD-Leu SD-Lue-Trp	√ ×
2	pBT3-N-LCD	pOst-Nub1	阳性对照	SD-Lue-Trp SD-Lue-Trp-His SD-Lue-Trp-His-Ade	√ √ √
3	pBT3-N-LCD	pPR3-N	阴性对照	SD-Lue-Trp SD-Lue-Trp-His SD-Lue-Trp-His-Ade	√ ×/少量 ×