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)"

[1] 林平, 姜玉梅, 陈瑛 . 几种油料作物中脂肪酸组成的研究及探讨. 江西科学, 2000,18(2):116-119.
doi: 10.3969/j.issn.1001-3679.2000.02.014
LIN P, JIANG Y M, CHEN Y . A study of the fatty acid content in several oil_bearing crops. JiangXi Science, 2000,18(2):116-119. (in Chinese)
doi: 10.3969/j.issn.1001-3679.2000.02.014
[2] BOLTON G E, SANDERS T H . Effect of roasting oil composition on the stability of roasted high-oleic peanuts. Journal of the American Oil Chemists' Society, 2002,79:129-132.
doi: 10.1007/s11746-002-0446-1
[3] CHANG S, PETERSON R J, Ho C T . Chemical reactions involved in the deep-fat frying of foods. Journal of the American Oil Chemists' Society, 1978,55(10):718-727.
doi: 10.1007/BF02665369
[4] 杜海, 郎春秀, 王伏林, 陈锦清, 吴关庭 . 油菜种子油酸含量的遗传改良. 核农学报, 2011,25(6):1179-1183.
DU H, LANG C X, WANG F L, CHEN J Q, WU G T . Genetic improvement of oleic acid content in rapeseed. Journal of Nuclear Agricultural Science, 2011,25(6):1179-1183. (in Chinese)
[5] SLEIGHT P . Cholesterol and coronary heart disease mortality. Australian & New Zealand Journal of Medicine, 1992,22(5):576-579.
doi: 10.1111/j.1445-5994.1992.tb00480.x pmid: 1449442
[6] SMITH D G, Song F, SHELDON T A . Cholesterol lowering and mortality: The importance of considering initial level of risk. British Medical Journal, 1993,306(6889):1367-1373.
doi: 10.1136/bmj.306.6893.1648 pmid: 8518602
[7] 王景梓, 徐贵发 . 单不饱和脂肪酸与冠心病的关系. 食品与药品, 2005,7(10A):21-23.
doi: 10.3969/j.issn.1672-979X.2005.10.007
WANG J Z, XU G F . Monounsaturated fatty acid in relation to coronary heart disease. Food and Drug, 2005,7(10A):21-23. (in Chinese)
doi: 10.3969/j.issn.1672-979X.2005.10.007
[8] 蒋秀琴, 刘立成, 赵福忠, 刘光前, 王旭 . 常见植物油脂肪酸含量的分析. 饲料博览, 2010(3):27-30.
doi: 10.3969/j.issn.1001-0084.2010.03.011
JIANG X Q, LIU L C, ZHAO G Z, LIU G Q, WANG X . Analysis of fatty acid in vegetable oils. Feed Review, 2010(3):27-30. (in Chinese)
doi: 10.3969/j.issn.1001-0084.2010.03.011
[9] 袁利文 . 植物油中主要脂肪酸含量的分析. 中国检验检测, 2018(1):18-21.
YUAN L W . Analysis of main fatty acid in vegetable oil. China Inspection Body and Laboratory, 2018(1):18-21. (in Chinese)
[10] 杨帆, 薛长勇 . 常用食用油的营养特点和作用研究进展. 中国食物与营养. 2013,19(3):63-66.
doi: 10.3969/j.issn.1006-9577.2013.03.015
YANG F, XUE C Y . Research advancement of nutritional characteristics and functions of common edible oils. Food and Nutrition in China, 2013,19(3):63-66. (in Chinese)
doi: 10.3969/j.issn.1006-9577.2013.03.015
[11] BROADWATER J A, AI J, LOEHR T M . Peroxodiferric intermediate of stearoyl-acyl carrier protein A9-desaturase: Oxidase reactivity during single turn over and implications for the mechanism of desaturation. Biochemistry, 1998,37:14664-14671.
doi: 10.1021/bi981839i
[12] ROUGHAN P G, SLACK C R . Cellular organization of glycerolipid metabolism. Annual Reviews Plant Physiology, 1982,33:97-132.
doi: 10.1146/annurev.pp.33.060182.000525
[13] DAMUDE H G, ZHANG H, FARRALL L, RIPP K G, TOMB J F, HOLLERBACH D, YADAV N S . Identification of bifunctional delta12/omega3 fatty acid desaturases for improving the ratio of omega3 to omega6 fatty acids in microbes and plants. Proceedings of the National Academy of Sciences of the United States of America, 2006,103(25):9446-9451.
doi: 10.1073/pnas.0511079103 pmid: 16763049
[14] PAUL R, REBECCA W, ANDREA C, REOBERT G U, LILIAN M . Effect of Δ9-stearoyl-ACP-desaturase-C mutants in a high oleic background on soybean seed oil composition. Theoretical and Applied Genetics, 2014,127(2):349-358.
doi: 10.1007/s00122-013-2223-5 pmid: 24193357
[15] ZHANG Y F, MAXIMOVA S N, GUILTINAN M J . Characterization of a stearoyl-acyl carrier protein desaturase gene from potential biofuel plant. Frontiers in Plant Science, 2015,6:239.
doi: 10.1016/j.gene.2014.03.047 pmid: 24680703
[16] BYFIELD G E, XUE H, UPCHURCH R G . Two genes from soybean encoding soluble Δ9 stearoyl-ACP desaturases. Crop Science, 2006,46(2):840-846.
doi: 10.2135/cropsci2005.06-0172
[17] FOFANA B, DUGUID S, CLOUTIER S . Cloning of fatty acid biosynthetic genes β-ketoacyl CoA synthase, fatty acid elongase, stearoyl-ACP desaturase, and fatty acid desaturase and analysis of expression in the early developmental stages of flax (Linum usitatissimum L.) seeds. Plant Science, 2004,166(6):1487-1496
[18] LIU Z, YANG X, FU Y . SAD, a stearoyl-acyl carrier protein desaturase highly expressed in high-oil maize inbred lines. Russian Journal of Plant Physiology, 2009,56(5):709-715.
doi: 10.1134/s1021443709050185
[19] SHANG X, CHENG C, DING J, GUO W . Identification of candidate genes from the SAD gene family in cotton for determination of cottonseed oil composition. Molecular Genetics and Genomics, 2017,292:173-186.
doi: 10.1007/s00438-016-1265-1
[20] KNUTZON D S, SCHERER D E, SCHRECHENGOST W E . Nucleotide sequence of a complementary DNA clone encoding stearoyl-ACP desaturase from castor bean, Ricinus communis. Plant Physiology, 1991,96:344-345
[21] KLINKENBERG J, FAIST H, SAUPE S, LAMBERTZ S, KRISCHKE M, STINGL N, FEKETE A, MUELLER M J, FEUSSNER I, HEDRICH R, DEEKEN R . Two fatty acid desaturases, stearoyl-acyl carrier protein Δ9- desaturase 6 and fatty acid desaturase 3 are involved in drought and hypoxia stress signaling in Arabidopsis crown galls. Plant Physiology, 2014,164:570-583.
[22] WENDY C, PAOLO L, NUNZIA S, MONICA D P, PAOLA S, VIRGINIA C, NOREEN M C, ALAN M M, PETER M, TONY A K, PHILIP J D, STEFANIA G, TEODORO C . Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance. Transgenic Research, 2008,17:769-782.
doi: 10.1007/s11248-008-9164-9 pmid: 18214708
[23] KNUTZON D S, THOMPSON G A, RADKE S E . Modification of brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene. Plant Biology, 1992,89:2624-2628.
doi: 10.1073/pnas.89.7.2624 pmid: 1557366
[24] WANG L, YU S, TONG C, ZHAO Y, LIU Y, SONG C, ZHANG Y, ZHANG X, WANG Y, HUA W, LI D, LI D, LI F, YU J, XU C, HAN X, HUANG S, TAI S, WANG J, XU X, LI Y, LIU S, VARSHNEY R, WANG J, ZHANG X . Genome sequencing of the high oil crop sesame provides insight into oil biosynthesis. Genome Biology, 2014,15(2):1-13.
doi: 10.1186/gb-2014-15-1-r1
[25] WANG L, YU J, LI D, ZHANG X . Sinbase: an integrated database to study genomics, genetics and comparative genomics in Sesamum indicum. Plant & Cell Physiology, 2015,56(1):e2.
doi: 10.1093/pcp/pcu175 pmid: 25480115
[26] 罗通, 邓骛远, 张富丽 . 植物硬脂酰-酰基载体蛋白脱饱和酶. 生命的化学, 2006,26(2):133-136.
doi: 10.3969/j.issn.1000-1336.2006.02.014
LUO T, DENG W Y, ZHANG F L . The stearoyl-acyl carrier protein desaturase in plants. Chemistry of Life, 2006,26(2):133-136. (in Chinese)
doi: 10.3969/j.issn.1000-1336.2006.02.014
[27] FOFANA B, CLOUTIER S, DUGUID S, CHING J, RAMPITSCH C . Gene expression of stearoyl-ACP desaturase and delta12 fatty acid desaturase 2 is modulated during seed development of flax (Linum usitatissimum). Lipids, 2006,41(7):705-712.
doi: 10.3923/ajps.2008.771.774 pmid: 17069354
[28] DU H, MIN H, HU J, LI J . Modification of the fatty acid composition in Arabidopsis and maize seeds using a stearoyl-acyl carrier protein desaturase-1(ZmSAD1) gene. BMC Plant Biology, 2016,16(1):137.
doi: 10.1186/s12870-016-0827-z pmid: 27297560
[29] AARDRA K, JOHN S, EDWARD W, LUDMILA L, DAVID H, PARDEEP K . The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the contribution of leaf isoforms to oleic acid synthesis. Plant Molecular Biology, 2007, 63, 2:257-271.
doi: 10.1007/s11103-006-9086-y pmid: 17072561
[30] LIU Q, SINGH S P, GREEN A G . High-stearic and high-oleic cottonseed oils produced by hpRNA-mediated post-transcriptional gene silencing. Plant Physiology, 2002,129:1732-1743.
doi: 10.1002/pssb.2221890110 pmid: 12177486
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