Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (10): 1678-1685.doi: 10.3864/j.issn.0578-1752.2019.10.002

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

Cloning and Functional Characterization of Sesame SiSAD Gene

ZHOU Rong1,LIU Pan1,LI DongHua1,ZHANG YanXin1,WANG LinHai1,ZHANG XiuRong1(),WEI Xin2()   

  1. 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, Wuhan 430062
    2 College of Life Sciences, Shanghai Normal University, Shanghai 200234
  • Received:2019-01-24 Accepted:2019-03-11 Online:2019-05-16 Published:2019-05-23
  • Contact: XiuRong ZHANG,Xin WEI E-mail:zhangxr@oilcrops.cn;xwei@shnu.edu.cn

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Abstract:

【Objective】 Sesame SiSAD (△9 stearoyl acyl-carrier-protein desaturase) gene was cloned and the expression of it was detected. It was transformed into Arabidopsis to investigate its role in the oleic acid synthesis. This study aims to provide molecular basis for the genetic improvement of sesame oleic acid content. 【Method】Total RNA was extracted from leaf of the variety Zhongzhi13 and then was reverse transcripted into cDNA. Using the primers that designed according to the reference genome, the coding region sequence of SiSAD was obtained by RT-PCR. The sequence was further compared with the reference genome. The conserved motifs of SiSAD protein were identified by InterPro and the homologous proteins of SiSAD were recognized by BLAST. A phylogenetic tree of SiSAD from sesame, Olea europaea var. sylvestris, Ipomoea nil, Ricinus communis, Lactuca sativa, Vitis vinifera, Citrus sinensis and Arabidopsis thaliana was constructed by neighbor-joining method to reveal the relationship of SiSAD protein in these species. Expression profiles of SiSAD in roots, stems, leaves, buds and seeds at two varieties Zhongzhi33 and Zhongfengzhi No.1 were investigated. The SiSAD gene was linked to a 35S vector and transformed into Arabidopsis by the Agrobacterium tumefaciens-mediated floral dip method. Based on the qRT-PCR detection, successful transformed Arabidopsis individuals were selected from the progenies. The stearic acid and oleic acid content in the seeds of transgenic T3 Arabidopsis seeds and Col-0 were detected and function of SiSAD was concluded. 【Result】 Total coding region sequence of SiSAD was cloned and the sequence was the same as the reference genome. It consisted of 1 152 nucleotides encoding a protein of 383 amino acids with a calculated molecular mass of 43 kD and a predicted pI of 6.18. We found that SiSAD gene contained one conserved function domain, which had been identified as a signature motif within the fatty acid desaturase family members. The similarity of SiSAD proteins from different species was quite high, indicating that SiSAD in different plant might had conserved function. The phylogenetic tree composed of SAD proteins showed that SiSAD, InSAD and OeSAD had been grouped together, suggested a close relationship of SiSAD protein among sesame, O. europaea var. sylvestris and Ipomoea nil. In contrast, SiSAD had a far relationship to AtSAD, CsSAD and RcSAD. qRT-PCR results showed that SiSAD is organ-specific expressed and had a highest expression level in seeds. We successfully constructed the overexpression vector of SiSAD and introduced the vector into Arabidopsis by Agrobacterium-mediated transformation. qRT-PCR was used to test the transcription of SiSAD in transgenic Arabidopsis plants. Compared with the Arabidopsis wild type Col-0, stearic acid content of 3 transgenic lines with overexpressed SiSAD gene was decreased by 3.0%, 4.8% and 6.1%, respectively. Which oleic acid content in these lines was increased by 2.8%, 4.3% and 7.8% (4.97% in average). 【Conclusion】 In this study, the total coding region sequence of SiSAD was cloned and function of SiSAD was characterized. SiSAD might plays important roles in improving oleic acid content, which could be used in the genetic improvement of oleic acid content in sesame seeds.

Key words: sesame, SiSAD, over-expression, oleic acid, functional characterization

Table 1

List of all PCR primers"

引物名称
Primer		 引物序列（5′-3′）
Primer sequence		 用途
SiSAD-F TCTAGAATGCAATCATCAGC CDS克隆
CDS Cloning
SiSAD-R GAGCTCCTACACAACTACTT
SISAF TGATGATTGAGAGGACAGT 实时荧光定量PCR
qRT-PCR
SISAR CGTTCTTGGAATGAAGTGTA
SIARactinF CCCGCTATGTATGTCGCCA
SIARactinR AACCCTCGTAGATTGGCACAG
PBISADF ACTCTAGAGGATCCCCGGGATGCAATCATCAGCATTC 植物表达载体构建
Constructs
PBISADR ATCGGGGAAATTCGAGCTCCTACACAACTACTTCCC
SiSAD-1F CATTGCCAACTTACCAGA 转基因鉴定引物
Transgenic line test
SiSAD-1R TCGCCTACGGCTATCA

Fig. 1

PCR amplified product of SiSAD in sesame"

Fig. 2

Putative conserved domain of SiSAD"

Fig. 3

Multiple alignment of deduced amino acid sequences of SADs"

Fig. 4

Phylogenetic tree analysis of SiSAD proteins from sesame and other plants"

Fig. 5

Expression profiles of SiSAD in different organs of Zhongfengzhi1 and Zhongzhi33"

Fig. 6

Expression profiles of SiSAD in transgenic Arabidopsis **: Significant difference (P<0.01). The same as below"

Fig. 7

Relative fatty acid content of transgenic Arabidopsis including SiSAD *: Significant difference (P<0.05)"

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