Identification and Preliminary Functional Verification of OsPUT1 Gene

doi:10.7668/hbnxb.20194136

Abstract

Abstract:

To explore the function and possible molecular mechanisms of amino acid transporters (AATs),firstly,a new hidden markov model was established to identify the members of the rice AAT family,and a phylogenetic tree was constructed to analyze the evolutionary relationship of AAT members between rice and Arabidopsis;secondly,the promoter,protein structure,and functional domain of rice polyamine transporter gene 1 (OsPUT1) were analyzed using bioinformatics methods;thirdly,the subcellular localization of OsPUT1 was determined by constructing the OsPUT1-GFP vector and expressing it in rice protoplasts;fourthly,the expression patterns of OsPUT1 gene in different tissues and under different stress conditions were detected using qRT-PCR;finally,the function of OsPUT1 gene was preliminarily studied using the OE,Nip and RNAi lines.The results showed that there were 96 OsAATs divided into 13 subfamilies in rice;the PUT subfamily contained 6 OsPUTs,among which OsPUT1 and AtPUT3 had the closest genetic distance and were distributed in the same branch.The promoter of OsPUT1 gene contained cis-acting elements related to growth and development,light regulation,plant hormones,and stress response;OsPUT1 protein contained the polyamine transporter domain PotE,and subcellular localization experiment indicated that it was located on the cell membrane;the expression of OsPUT1 gene was relatively high in leaves,while was low in flowers;gene expression was inhibited by JA,mannitol,and ABA,while decreased and then returned to normal under low temperature stress,yet increased and then decreased under SA,Spm and paraquat treatment,however,under sodium chloride treatment,the gene expression level first increased,reached its peak at 1 h,then decreased,reached its lowest at 12 h,and returned to normal levels at 24 h;OE lines significantly reduced resistance to paraquat,while RNAi lines significantly increased resistance to paraquat.Overall,the OsPUT1 protein might have the function of transporting PA and participating in stress response.

Key words: Rice, Polyamine transporters, Subcellular localization, Amino acids, Amino acid transporters, Polyamines

ZHANG Bin, LI Xiaohui. Identification and Preliminary Functional Verification of OsPUT1 Gene[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5): 10-18. doi: 10.7668/hbnxb.20194136.

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Figures/Tables 6

Tab.1 Primer sequences

引物名称 Primer name	序列(5'-3') Sequence(5'-3')	用途 Usage
GFP-F	AGGGTACCCGGGGATCCTCTAGAATGCGCGCTTCCACTCCGCA	亚细胞定位
GFP-R	CATGGTACTAGTGTCGACTCTAGACGGAACCAACGGCTCGTCCG
RNAi-F	CCAGAAACCCACGTCATGCCAGTT	RNAi株系检测
RNAi-R	GCGGTCTGCACCATCGTCAACC
OE-F	GCCTCGCTCCAGTCAATGAC	OE株系检测
OE-R	ATTCCGGAAGTGCTTGACAT
Q-Actin-F	AGCTATCGTCCACAGGAA	内参基因
Q-Actin-R	ACCGGAGCTAATCAGAGT
Q-PUT1-F	CAAGAGAAGCCCACCGTCGAGA	qRT-PCR
Q-PUT1-R	AAGATGAGCGGGATCATCGAAACC

Fig.1 Phylogenetic analysis of AAT family in rice and Arabidopsis

Fig.2 Protein structure,cis-acting elements and subcellular localization of OsPUT1 gene A.OsPUT1 protein structure:Black bar.The length of amino acid sequence,White bars.PotE structural domains; B.cis-acting element of OsPUT1 gene;C.Subcellular localization of OsPUT1 protein.

Fig.3 Expression pattern of OsPUT1 gene Different lowercase letters indicate significant difference(P<0.05),and the error line represents the standard deviation.The same as Fig.4-5.

Fig.4 Identification of transgenic seedlings and screening of homozygous lines A.DNA level detection of RNAi lines:1-8.Resistant seedlings,9.Positive control;B.DNA level detection of OE lines:1-7.Resistant seedlings, 8.Positive control;C.RNA level detection of OE lines;D.RNA level detection of RNAi lines;E.T₂ seed screening;F.T₃ seed screening.

Fig.5 Growth of rice seedlings under paraquat stress A.Phenotype of Nip lines cultured for 10 days(0.2 μmol/L paraquat);B.The root length of Nip lines cultured for 10 days;C.Phenotypes of OE,Nip and RNAi lines cultured for 10 days(0.2 μmol/L paraquat);D.Phenotypes of OE,Nip and RNAi lines cultured for 10 days(0 μmol/L paraquat);E.The number of roots of OE,Nip and RNAi lines cultured for 10 days;F.The plant height of OE,Nip and RNAi lines cultured for 10 days;G.Root length of OE,Nip and RNAi lines cultured for 10 days;H.Proline content in roots of OE,Nip and RNAi lines cultured for 10 days;I.Root vitality of OE,Nip and RNAi lines cultured for 16 days(0.2 μmol/L paraquat).

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