Cloning of Gene MdSGR2 from Apple and Construction of Plant Overexpression Vector

doi:10.7668/hbnxb.20192675

Abstract

Abstract:

Gene STAYGREEN(SGR)plays an important role in the process of plant chlorophyll degradation and metabolism.To explore the biological function of gene SGR2,MdSGR2 gene sequence was cloned from the peel of Granny Smith by RT-PCR.The homology,physicochemical properties,protein structure and cis-acting elements of the promoter were predicted and analyzed by bioinformatics software.The plant overexpression vector was constructed by double enzyme digestion.The results showed that the complete open reading frame(ORF)cDNA of gene MdSGR2 was 840 bp.Encoding 279 amino acids in total,it belonged to the Staygreen superfamily.Physicochemical analysis showed that the total molecular weight of the protein was 31.27 ku,and the isoelectric point pI was 8.52,indicating that the protein was an unstable hydrophilic protein.Homology analysis showed that MdSGR2 encoded amino acid sequence was of the highest homology with Pyrus ussuriensis,reaching 84.12%.The protein structure analysis showed that the secondary structure and tertiary structure of MdSGR2 was determined structurally to be 40.55% α-helix and 44.44% random coil.Promoter analysis showed that the promoter region of the gene contained cryogenic cis-acting elements,photo-responsive elements and induced cis-acting elements such as abscisic acid,salicylic acid and methyl jasmonate response elements.These results suggest that gene MdSGR2 may be regulated by many factors such as environment and hormones.The plant overexpression vector pCAMBIA2301-MdSGR2 was successfully constructed.

Key words: Apple, MdSGR2, Gene cloning, Bioinformatics analysis, Overexpression vector

ZHAO Huiying, YANG Guangkai, GAO Yan, ZHANG Xiaojun, HAO Yanyan. Cloning of Gene MdSGR2 from Apple and Construction of Plant Overexpression Vector[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(2): 42-48. doi: 10.7668/hbnxb.20192675.

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Figures/Tables 6

Fig.1 PCR amplification of gene MdSGR2
M.D2000 DNA Marker;1—4.PCR products of MdSGR2.

Fig.2 Homologous amino acid sequence alignment of gene MdSGR2
Md.Malus domestica;Pu.Pyrus ussuriensis;Pp.Prunus persica;Pa.Prunus avium;Fa.Fragaria vesca subsp.vesca.

Fig.3 Phylogenetic analysis of MdSGR2 protein and other homologous proteins

Fig.4 Tertiary structure model of MdSGR2 protein

Tab.1 Cis-active elements and functions in the promoter region of MdSGR2

顺式作用元件 Cis-acting element	序列 Sequence	功能 Function	数量 Number
ABRE	CACGTG/ACGTG	脱落酸响应顺式作用元件	5
ACE	CTAACGTATT	光响应顺式作用元件	1
CGTCA-motif/TGACG-motif	CGTCA/TGACG	茉莉酸甲酯响应顺式调节元件	4
G-Box	CACGTG/CACGTT/CACGAC/ACACGTGT	光响应顺式调节元件	7
LTR	CCGAAA	低温响应顺式作用元件	2
TCA-element	CCATCTTTTT	水杨酸响应顺式作用元件	1

Fig.5 Construction of pCAMBIA2301-MdSGR2 overexpression vector
A.Double enzyme-digested of pMD-MdSGR2;B.Double enzyme-digested of vector pCAMBIA2301;C.Results of identification of overexpression vector pCAMBIA2301-MdSGR2.M.D2000 DNA Marker;1.Double digestion product of pMD-MdSGR2 plasmid;2.The double digestion product of vector pCAMBIA2301;3.Double enzyme-digested product of pCAMBIA2301-MdSGR2.

References 34

[1]	迟馨,王心悦,宋来庆,姜中武,杜晓云.苹果果皮着色的研究进展[J].烟台果树, 2019(3):4-6.doi:10.3969/j.issn.1005-9938.2019.03.003.
	Chi X,Wang X Y,Song L Q,Jiang Z W,Du X Y.Research progress of apple peel coloring[J].Yantai Fruits,2019(3):4-6.
[2]	Lancaster J E,Dougall D K.Regulation of skin color in apples[J].Critical Reviews in Plant Sciences,1992,10(6):487-502.doi:10.1080/07352689209382324.
[3]	魏珑. 果蔬叶绿素降解机理研究进展[J].食品安全导刊, 2016(9):49-50.doi:10.16043/j.cnki.cfs.2016.09.024.
	Wei L.Research progress on chlorophyll degradation mechanism of fruits and vegetables[J].China Food Safety Magazine,2016(9):49-50.
[4]	Gómez-Lobato M E,Hasperué J H,Civello P M,Chaves A R,Martínez G A.Effect of 1-MCP on the expression of chlorophyll degrading genes during senescence of broccoli(Brassica oleracea L.)[J].Scientia Horticulturae,2012,144:208-211.doi:10.1016/j.scienta.2012.07.017.
[5]	Hörtensteiner S,Kräutler B.Chlorophyll breakdown in higher plants[J].Biochimica et Biophysica Acta(BBA)-Bioenergetics,2011,1807(8):977-988.doi:10.1016/j.bbabio.2010.12.007.
[6]	董笛,张宇昕,刘畅,王焕英,李殷睿智,刘卓成,王梦迪,晁跃辉,韩烈保.日本结缕草ZjNOL基因的克隆及转录自激活检测[J].中国草地学报,2019,41(3):15-21.doi:10.16742/j.zgcdxb.20180140.
	Dong D,Zhang Y X,Liu C,Wang H Y,Li Y R Z,Liu Z C,Wang M D,Chao Y H,Han L B.Cloning and autonomous transcriptional activation testing of ZjNOL(NYC1-like)in Zoysia japonica[J].Chinese Journal of Grassland,2019,41(3):15-21.
[7]	刘建新,王国夫,许骅,丁华侨.姜荷花叶绿素降解酶基因PAO的分离及特征[J].绍兴文理学院学报(自然科学),2020,40(2):42-48,2.doi:10.16169/j.issn.1008-293x.K.2020.04.006.
	Liu J X,Wang G F,Xu H,Ding H Q.Isolation and characteristics of chlorophyll degrading gene PAO from Curcuma alismatifolia[J].Journal of Shaoxing University(Natural Science),2020,40(2):42-48,2.
[8]	刘宽亮,赵志常,罗睿雄,陈业渊.杧果MiPAO基因的克隆表达分析及其基因沉默载体的构建[J].华北农学报,2018,33(S1):1-9.doi:10.7668/hbnxb.2018.S1.001.
	Liu K L,Zhao Z C,Luo R X,Chen Y Y.Cloning and expression analysis of pheophorbide a oxygenase gene(MiPAO)and construction of silencing expression vector from mango[J].Acta Agriculturae Boreali-Sinica,2018,33(S1):1-9.
[9]	刘宽亮,赵志常,高爱平,黄建峰,党志国,罗睿雄,陈业渊.芒果(Mangifera indica)RCCR基因的克隆及其基因沉默载体的构建[J].分子植物育种,2018,16(10):3174-3181.doi:10.13271/j.mpb.016.003174.
	Liu K L,Zhao Z C,Gao A P,Huang J F,Dang Z G,Luo R X,Chen Y Y.Cloning RCCR gene from mango(Mangifera indica)and the construction of gene silencing vector[J].Molecular Plant Breeding,2018,16(10):3174-3181.
[10]	Sakuraba Y,Park S Y,Paek N C.The divergent roles of STAYGREEN(SGR)homologs in chlorophyll degradation[J].Molecules and Cells,2015,38(5):390-395.doi:10.14348/molcells.2015.0039.
[11]	Armstead I,Donnison I,Aubry S,Harper J,Hörtensteiner S,James C,Mani J,Moffet M,Ougham H,Roberts L,Thomas A,Weeden N,Thomas H,King I.Cross-species identification of Mendel's I locus[J].Science,2007,315(5808):73.doi:10.1126/science.1132912.
[12]	Park S Y,Yu J W,Park J S,Li J J,Yoo S C,Lee N Y,Lee S K,Jeong S W,Seo H S,Koh H J,Jeon J S,Park Y I,Paek N C.The senescence-induced staygreen protein regulates chlorophyll degradation[J].The Plant Cell,2007,19(5):1649-1664.doi:10.1105/tpc.106.044891.
[13]	Ren G D,An K,Liao Y,Zhou X,Cao Y J,Zhao H F,Ge X C,Kuai B K.Identification of a novel chloroplast protein AtNYE1 regulating chlorophyll degradation during leaf senescence in Arabidopsis[J].Plant Physiology,2007,144(3):1429-1441.doi:10.1104/pp.107.100172.
[14]	Barry C S,McQuinn R P,Chung M Y,Besuden A,Giovannoni J J.Amino acid substitutions in homologs of the STAY-GREEN protein are responsible for the green-flesh and chlorophyll retainer mutations of tomato and pepper[J].Plant Physiology,2008,147(1):179-187.doi:10.1104/pp.108.118430.
[15]	方永丰,李永生,白江平,慕平,孟亚雄,张金林,王汉宁,尚勋武.玉米持绿相关QTL整合图谱构建及一致性QTL区域内候选基因发掘[J].草业学报,2012,21(4):175-185.
	Fang Y F,Li Y S,Bai J P,Mu P,Meng Y X,Zhang J L,Wang H N,Shang X W.Construction of integration QTL map and identification of candidate genes for stay-green in maize[J].Acta Prataculturae Sinica,2012,21(4):175-185.
[16]	Rong H,Tang Y Y,Zhang H,Wu P Z,Chen Y P,Li M R,Wu G J,Jiang H W.The Stay-Green Rice like(SGRL)gene regulates chlorophyll degradation in rice[J].Journal of Plant Physiology,2013,170(15):1367-1373.doi:10.1016/j.jplph.2013.05.016.
[17]	任钧,王晓磊,高炯,周强,徐永平,蒯本科.国内大豆品种资源中滞绿(stay-green)性状的初步研究[J].植物生理学报,2014,50(9):1336-1346.doi:10.13592/j.cnki.ppj.2014.1020.
	Ren J,Wang X L,Gao J,Zhou Q,Xu Y P,Kuai B K.A preliminary study on the stay-green traits of soybean germplasm varieties[J].Plant Physiology Journal,2014,50(9):1336-1346.
[18]	Nakano M,Yamada T,Masuda Y,Sato Y,Kobayashi H,Ueda H,Morita R,Nishimura M,Kitamura K,Kusaba M.A green-Cotyledon/stay-green mutant exemplifies the ancient whole-genome duplications in soybean[J].Plant and Cell Physiology,2014,55(10):1763-1771.doi:10.1093/pcp/pcu107.
[19]	Zhu K J,Zheng X J,Ye J L,Huang Y,Chen H Y,Mei X H,Xie Z Z,Cao L X,Zeng Y L,Larkin R M,Xu Q,Perez-Roman E,Talòn M,Zumajo-Cardona C,Wurtzel E T,Deng X X.Regulation of carotenoid and chlorophyll pools in hesperidia,anatomically unique fruits found only in Citrus[J].Plant Physiology,2021,187(2):829-845.doi:10.1093/plphys/kiab291.
[20]	Sakuraba Y,Park S Y,Kim Y S,Wang S H,Yoo S C,Hörtensteiner S,Paek N C.Arabidopsis STAY-GREEN2 is a negative regulator of chlorophyll degradation during leaf senescence[J].Molecular Plant,2014,7(8):1288-1302.doi:10.1093/mp/ssu045.
[21]	Luo Z D,Zhang J H,Li J H,Yang C X,Wang T T,Ouyang B,Li H X,Giovannoni J,Ye Z B.A STAY-GREEN protein SlSGR1 regulates lycopene and β-carotene accumulation by interacting directly with SlPSY1 during ripening processes in tomato[J].New Phytologist,2013,198(2):442-452.doi:10.1111/nph.12175.
[22]	Hu Z L,Deng L,Yan B,Pan Y,Luo M,Chen X Q,Hu T Z,Chen G P.Silencing of the LeSGR1 gene in tomato inhibits chlorophyll degradation and exhibits a stay-green phenotype[J].Biologia Plantarum,2011,55(1):27-34.doi:10.1007/s10535-011-0004-z.
[23]	Sakuraba Y,Schelbert S,Park S Y,Han S H,Lee B D,Andrès C B,Kessler F,Hörtensteiner S,Paek N C.STAY-GREEN and chlorophyll catabolic enzymes interact at light-harvesting complex Ⅱ for chlorophyll detoxification during leaf senescence in Arabidopsis[J].The Plant Cell,2012,24(2):507-518.doi:10.1105/tpc.111.089474.
[24]	田雪婷,吴晗笑,王雷,田晓宁,李瑞,戚英伟,任小林.1-MCP处理对采后澳洲青苹苹果叶绿素降解的影响[J].果树学报,2020,37(5):734-742.doi:10.13925/j.cnki.gsxb.20190595.
	Tian X T,Wu H X,Wang L,Tian X N,Li R,Qi Y W,Ren X L.Effect of 1-MCP treatment on chlorophyll degradation in postharvest Granny Smith apple fruits[J].Journal of Fruit Science,2020,37(5):734-742.
[25]	宋小青,任亚梅,张艳宜,师俊玲,樊明涛.采后猕猴桃叶绿素降解机制及1-MCP处理对其代谢的影响[J].食品科学,2017,38(17):260-265.doi:10.7506/spkx1002-6630-201717042.
	Song X Q,Ren Y M,Zhang Y Y,Shi J L,Fan M T.Mechanism of chlorophyll degradation and effect of 1-MCP treatment on chlorophyll metabolism in postharvest kiwifruit[J].Food Science,2017,38(17):260-265.
[26]	刘凌云,檀鹏辉,姜红岩,常智慧,滕珂.过表达青绿苔草滞绿基因CbSGR加速拟南芥叶绿素降解和衰老[J].中国草地学报,2020,42(4):15-24.doi:10.16742/j.zgcdxb.20190314.
	Liu L Y,Tan P H,Jiang H Y,Chang Z H,Teng K.Overexpression of CbSGR,A stay-green gene from Carex breviculmis accelerated chlorophyll degradation and senescence in Arabidopsis thaliana[J].Chinese Journal of Grassland,2020,42(4):15-24.
[27]	罗志丹. 番茄果实颜色与质地调控基因SlSGR1和nsLTP的功能鉴定[D].武汉:华中农业大学,2012.
	Luo Z D.Functiolal analysis of fruit color and texture regulated genes SlSGR1 and nsLTP in tomato[D].Wuhan:Huazhong Agricultural University,2012.
[28]	张敬. 多年生黑麦草滞绿基因STAY-GREEN的功能研究[D].南京:南京农业大学,2017.
	Zhang J.Functional characterization of STAY-GREEN gene in Perennial ryegrass[D].Nanjing:Nanjing Agricultural University,2017.
[29]	Teng K,Chang Z H,Xiao G Z,Guo W E,Xu L X,Chao Y H,Han L B.Molecular cloning and characterization of a chlorophyll degradation regulatory gene(ZjSGR)from Zoysia japonica[J].Genetics and Molecular Research,2016,15(2):15028176.doi:10.4238/gmr.15028176.
[30]	张倩丽. 基于TRV-VIGS的菊花脑滞绿基因CnSGR1功能鉴定[D].南京:南京农业大学,2017.
	Zhang Q L.Functional identification of Chrysanthemum nankingense stay green gene CnSGR1 based on TRV-VIGS[D].Nanjing:Nanjing Agricultural University,2017.
[31]	Yang M M,Zhu S B,Jiao B Z,Duan M,Meng Q W,Ma N N,Lü W.SlSGRL,a tomato SGR-like protein,promotes chlorophyll degradation downstream of the ABA signaling pathway[J].Plant Physiology and Biochemistry,2020,157:316-327.doi:10.1016/j.plaphy.2020.10.028.
[32]	Tan X L,Fan Z Q,Shan W,Yin X R,Kuang J F,Lu W J,Chen J Y.Association of BrERF72 with methyl jasmonate-induced leaf senescence of Chinese flowering cabbage through activating JA biosynthesis-related genes[J].Horticulture Research,2018,5:22.doi:10.1038/s41438-018-0028-z.
[33]	檀鹏辉,袁丽丽,樊波,于安东,董笛,滕珂,晁跃辉.日本结缕草滞绿基因ZjSGR对烟草的转化及功能分析[J].草业学报,2017,26(5):155-162.doi:10.11686/cyxb2016492.
	Tan P H,Yuan L L,Fan B,Yu A D,Dong D,Teng K,Chao Y H.Functional analysis of thestay-green gene ZjSGR from Zoysia japonica using transgenic Nicotiana tabacum[J].Acta Prataculturae Sinica,2017,26(5):155-162.
[34]	周志湘,韩烈保,晁跃辉,张铁军.匍匐剪股颖ZjSGR基因的遗传转化及生理生化分析[J].草业科学,2020,37(3):444-450.doi:10.11829/j.issn.1001-0629.2019-0600.
	Zhou Z X,Han L B,Chao Y H,Zhang T J.Screening and analysis of ZjSGR transgenic creeping bentgrass(Agrostis stolonifera)[J].Pratacultural Science,2020,37(3):444-450.

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