Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (17): 3432-3442.doi: 10.3864/j.issn.0578-1752.2020.17.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Function Analysis of Sesame Galactinol Synthase Gene SiGolS6 in Arabidopsis

LIU AiLi(),WEI MengYuan,LI DongHua,ZHOU Rong,ZHANG XiuRong,YOU Jun()   

  1. Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs, Wuhan 430062
  • Received:2019-12-30 Accepted:2020-03-08 Online:2020-09-01 Published:2020-09-11
  • Contact: Jun YOU E-mail:liuailihappy@126.com;junyou@caas.cn

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Abstract:

【Objective】SiGolS6 is a member of galactinol synthase family in sesame, which may play important role in drought stress resistance. Studying the function of SiGolS6 in plant drought stress resistance, so as to provide theoretical basis and gene resources for drought resistance improvement of sesame. 【Method】The galactinol synthase gene, SiGolS6, was isolated from sesame through reverse transcription PCR (RT-PCR). The sequence was analyzed by bioinformatics tools such as InterProScan, ClustalX2 and MEGA5.2. Then the function of SiGolS6 in plant drought resistance was characterized by phenotype analysis and physiological index measurement of SiGolS6 transgenic Arabidopsis thaliana. 【Result】The total length of the CDS sequence of SiGolS6 was 921 bp, encoding a polypeptide of 306 amino acids. The molecular weight of SiGolS6 is 35.07 kD and its isoelectric point is 4.7. Sequence analysis showed that SiGolS6 protein contained conserved glycosyl transferase domain (IPR002495), belonging to the glycosyl transferase superfamily. Based on phylogenetic tree constructed with GolS proteins from sesame and other species, SiGolS6 was highly similar to the homologous genes in potato. Six independent transgenic Arabidopsis thaliana lines were identified by hygromycin screening and PCR. Three transgenic lines (OE-2, OE-3, and OE-4) with high expression levels were identified by qRT-PCR, and used for subsequent experiment. Raffinose content in those transgenic plants was higher than that in wild-type (WT) plants. Under drought stress, the wilting degree of transgenic lines was less than that of WT lines. After 28 d of drought stress and 5 d of recovery, the fresh weight of transgenic lines was significantly higher than that of WT, but there was no significant difference under normal conditions. After 5 d of recovery, 50% of transgenic plants recovered, while the survival rate of WT was less than 10%. After 21 d of drought stress, the relative electrical conductivity, ROS accumulation and MDA content of transgenic plants were significantly lower than those of WT, while the relative activities of SOD and POD were significantly higher than those of WT.【Conclusion】Overexpression of SiGolS6 in Arabidopsis thaliana could improve the drought tolerance of transgenic plant.

Key words: sesame, galactinol synthase, transgenic Arabidopsis, drought tolerance, functional analysis

Table 1

The primers used in the study"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)
SiGolS6q-F CCAATCCCCGCAACCTATAAC
SiGolS6q-R TGCTCTTTGTCGGTGTACTTC
Actinq-F CCCGCTATGTATGTCGCCA
Actinq-R AACCCTCGTAGATTGGCACAG
GolS6FL-F GGGGTACCATGGTTCCTGAAATTATCATTCC
GolS6FL-R CGGGATCCTCAACCGGAAACTTGATCAATTG

Fig. 1

Multiple sequence alignment (A) and phylogenetic analysis (B) of SiGolS6 and its homologs"

Fig. 2

Acquisition and identification of transgenic Arabidopsis A, B: Transgenic (A) and wild type (B) plants growing on selective medium containing hygromycin; C: Identification of positive T2 generation transgenic Arabidopsis plants; 1-6: SiGolS6 transgenic lines; WT: Wild type; D: Transcript level of SiGolS6 in transgenic Arabidopsis plants. * indicate significant difference at P<0.05 level; **indicates significant difference at P<0.01 level. The same as below"

Fig. 3

Raffinose content in transgenic Arabidopsis plants"

Fig. 4

Characteristics of transgenic plants under drought stress A: Phenotype of WT and transgenic plants under drought stress; B: Water loss from detached leaves of WT and transgenic plants at indicated time points; C: Survival rate of WT and transgenic plants after drought stress; D-E: Fresh weight (D) and relative electrical conductivity (E) of WT and transgenic plants after drought stress"

Fig. 5

Determination of oxidation-related physiological indexes in transgenic Arabidopsis plants A: Nitro blue tetrazolium staining of leaves from WT and transgenic plants under normal or drought treatments; B-D: Relative content of MDA (B), relative activity of SOD (C) and relative activity of POD (D) of WT and transgenic plants after drought stress"

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