百合鳞茎发育过程中淀粉合成相关酶基因的克隆及表达分析
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摘要
该研究通过同源克隆技术克隆腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)、颗粒结合淀粉合酶(GBSS)和可溶性淀粉合酶(SSS) 3类百合淀粉合成关键酶基因,分析这三类淀粉合成关键酶基因的表达变化,测定百合鳞茎膨大发育中淀粉含量变化。结果表明:(1) AGPase具有GlgC家族蛋白PLN02241蛋白结构特征及cl11394家族蛋白ADP_Glucose_PP与NTP_transferase结构域,获登录号KP751443; GBSS与SSS具有cl10013家族蛋白Glyco_transf_5,GT1_Glycogen_synthase_DULL1_like结构域,获登录号分别为KP751444、KP751445。(2)百合鳞茎形成与膨大发育过程中,淀粉含量呈现递增趋势,鳞茎盘开始分化茎杆时其淀粉含量最高,达到44.52%。鳞茎与叶片部位的三个淀粉合成相关酶基因表达量均逐渐增加;在鳞茎膨大后茎杆分化阶段,三个淀粉合成相关酶基因表达量达到最高,AGPase、GBSS、SSS在鳞片中的表达量分别为10.79,6.92和5.12,叶片中的表达量分别为6.79,5.22和4.41,鳞片中的表达量大幅度高于叶片;淀粉合成相关酶基因的表达量变化与淀粉含量、鳞茎的膨大发育成正相关。这为鳞茎的繁殖生产提供了可通过调节淀粉合成关键酶基因表达促进百合鳞茎膨大发育的思路。
The key enzyme genes of lily starch synthesis,ADP glucose pyrophosphorylase (AGPase),granule binding starch synthase (GBSS) and soluble starch synthase (SSS),were cloned by homologous cloning,and the bioinformatics analysis was performed. The bulblets and leaves of lily during the four stages of bulblet swelling development were used to determine the starch content in bulblets at different stages,and the expression of the key genes encoding starch synthesis-related enzymes in the process of bulblet swelling development were analyzed using fluorescence quantitative PCR. The results were as follows:(1) AGPase had PLN02241 protein domain belonging to Glg C family protein and ADP_Glucose_PP and NTP_transferase domain belonging to cl11394 family protein. The accession number of AGPase gene was KP751443. GBSS and SSS had Glyco_transf_5,GT1_Glycogen_synthase_DULL1_like conserved domain belonging to cl10013 family protein. The accession numbers of GBSS and SSS genes were KP751445 and KP751444,respectively. (2) The starch content showed an increasing trend in the process of the formation and development of lily bulblet swelling. The starch content was up to 44.52% at the stem differentiation stage after bulblet swelling,which was the highest. The three starch synthesis-related genes were up-regulated gradually in bulblets and leaves. The expression of these three starch synthesis-related genes were up to the peak at the stem differentiation stage after bulblet swelling. The expression of AGPase,GBSS and SSS were 10.79,6.92 and 5.12 in bulblet,and were 6.79,5.22 and 4.41 in leaves,respectively. The genes expression were much higher in bulblets than in leaves. There was a positive correlation between the starch synthesis-related gene expression and the content of starch and bulblet swelling. The finding provides a reference for propagation production of lily bulblet,by which the expression of key synthesis-related genes can be regulated to promote the swelling development of lily bulblet.
The key enzyme genes of lily starch synthesis,ADP glucose pyrophosphorylase (AGPase),granule binding starch synthase (GBSS) and soluble starch synthase (SSS),were cloned by homologous cloning,and the bioinformatics analysis was performed. The bulblets and leaves of lily during the four stages of bulblet swelling development were used to determine the starch content in bulblets at different stages,and the expression of the key genes encoding starch synthesis-related enzymes in the process of bulblet swelling development were analyzed using fluorescence quantitative PCR. The results were as follows:(1) AGPase had PLN02241 protein domain belonging to Glg C family protein and ADP_Glucose_PP and NTP_transferase domain belonging to cl11394 family protein. The accession number of AGPase gene was KP751443. GBSS and SSS had Glyco_transf_5,GT1_Glycogen_synthase_DULL1_like conserved domain belonging to cl10013 family protein. The accession numbers of GBSS and SSS genes were KP751445 and KP751444,respectively. (2) The starch content showed an increasing trend in the process of the formation and development of lily bulblet swelling. The starch content was up to 44.52% at the stem differentiation stage after bulblet swelling,which was the highest. The three starch synthesis-related genes were up-regulated gradually in bulblets and leaves. The expression of these three starch synthesis-related genes were up to the peak at the stem differentiation stage after bulblet swelling. The expression of AGPase,GBSS and SSS were 10.79,6.92 and 5.12 in bulblet,and were 6.79,5.22 and 4.41 in leaves,respectively. The genes expression were much higher in bulblets than in leaves. There was a positive correlation between the starch synthesis-related gene expression and the content of starch and bulblet swelling. The finding provides a reference for propagation production of lily bulblet,by which the expression of key synthesis-related genes can be regulated to promote the swelling development of lily bulblet.
引文
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YANG PP,XU LF,XU H,et al.,2017.Histological and transcriptomic analysis during bulbil formation in Lilium lancifolium[J].Front Plant Sci,8:1508.
ZHANG JZ,FAN YP,YU RC,et al.,2014.Regeneration of bulblets from bulb scale explants of Lanzhou lily(Lilium davidii var.unicolor)and enhancement of bulblet growth[J].Propag Ornam Plants,14(3):112-116.
ZHANG JZ,WEI SL,SUN JM,et al.,2016.Bulblet formation and development of Lanzhou lily(Lilium davidii var.unicolor)by tissue culture[J].Guihaia,36(3):297-302.[张进忠,韦绍龙,孙嘉曼,等,2016.兰州百合组培鳞茎发育研究[J].广西植物,36(3):297-302.]
ZHANG YN,LI ZT,ZHANG YB,et al.,2016.Culture optimization of bulb formation and swelling in vitro of Lilium dauricum[J].Jiangsu Agric Sci,44(4):74-78.[张彦妮,李兆婷,张艳波,等,2016.毛百合试管鳞茎形成和膨大的培养优化[J].江苏农业科学,44(4):74-78.]
ZHAO J,ZHAO ZG,TANG FL,et al.,2017.Progress in Lilium brownie var.viridulum[J].Guizhou Agric Sci,45(7):78-81.[赵健,赵志国,唐凤鸾,等,2017.龙牙百合的研究进展[J].贵州农业科学,45(7):78-81.]
GAO ZY,HUANG DN,1998.A key enzyme for the synthesis of sucrose and starch in plantsⅠ.Adenosine diphosphoglucose pyrophosphprylase[J].Chem Life,(3):28-31.[高振宇,黄大年,1998.植物中合成蔗糖和淀粉的关键酶Ⅰ.腺苷二磷酸-葡萄糖焦磷酸化酶[J].生命的化学,(3):28-31.]
GUO Y,DONG L,LIU C,2012.The relationship between development of bulblet and changes of carbohydrates of pot lily[J].Henan Agric Sci,41(5):117-120.[郭燕,董丽,刘春,2012.盆栽百合鳞茎发育与碳水化合物变化的关系[J].河南农业科学,41(5):117-120.]
LIU XH,ZHENG LX,ZHENG LM,et al.,2013.Determination of amylose and amylopectin in the commonly used starch materials by dual-wavelength spectrophotometry[J].Guangdong Agric Sci,40(18):97-100.[刘襄河,郑丽璇,郑丽勉,等,2013.双波长法测定常用淀粉原料中直链淀粉、支链淀粉及总淀粉含量[J].广东农业科学,40(18):97-100.]
MIAO HX,SUN PG,ZHANG KX,et al.,2016.Research progress on expression regulation mechanism of genes encoding granule-bound starch synthase in plants[J].Biotechnol Bull,32(3):18-23.[苗红霞,孙佩光,张凯星,等,2016.植物颗粒结合淀粉合成酶(GBSS)基因的表达调控机制研究进展[J].生物技术通报,32(3):18-23.]
QIN XH,CUI XL,CHEN XH,et al.,2015.Effect of different factors on bulblet swelling in tissue culture of Lanzhou lily[J].For Sci Technol,(12):44-47.[秦新惠,崔兴林,陈学红,等,2015.不同因子对兰州百合组培小鳞茎膨大的影响研究[J].林业科技通讯,(12):44-47.]
QIU S,TANG FL,XIA K,et al.,2017.Propagation culture and bulbs development of Lilium brownii var.viridulum[J].Guangdong Agric Sci,44(4):61-66.[仇硕,唐凤鸾,夏科,等,2017.龙牙百合增殖培养及鳞茎生长发育研究[J].广东农业科学,44(4):61-66.]
RAJEEVAN MS,RANAMUKHAARACHCHI DG,VERNONSD,et al.,2001.Use of real-time quantitative PCR to validate the results of c DNA array and differential display PCRtechnologies[J].Methods,25(4):443-451.
WANG Q,SUN WJ,BAO Y,2017.Evolutionary pattern of the GBSS gene family in plants[J].Acta Bot Sin,52(2):179-187.[王倩,孙文静,包颖,2017.植物颗粒结合淀粉合酶GBSS基因家族的进化[J].植物学报,52(2):179-187.]
YANG PP,XU LF,XU H,et al.,2017.Histological and transcriptomic analysis during bulbil formation in Lilium lancifolium[J].Front Plant Sci,8:1508.
ZHANG JZ,FAN YP,YU RC,et al.,2014.Regeneration of bulblets from bulb scale explants of Lanzhou lily(Lilium davidii var.unicolor)and enhancement of bulblet growth[J].Propag Ornam Plants,14(3):112-116.
ZHANG JZ,WEI SL,SUN JM,et al.,2016.Bulblet formation and development of Lanzhou lily(Lilium davidii var.unicolor)by tissue culture[J].Guihaia,36(3):297-302.[张进忠,韦绍龙,孙嘉曼,等,2016.兰州百合组培鳞茎发育研究[J].广西植物,36(3):297-302.]
ZHANG YN,LI ZT,ZHANG YB,et al.,2016.Culture optimization of bulb formation and swelling in vitro of Lilium dauricum[J].Jiangsu Agric Sci,44(4):74-78.[张彦妮,李兆婷,张艳波,等,2016.毛百合试管鳞茎形成和膨大的培养优化[J].江苏农业科学,44(4):74-78.]
ZHAO J,ZHAO ZG,TANG FL,et al.,2017.Progress in Lilium brownie var.viridulum[J].Guizhou Agric Sci,45(7):78-81.[赵健,赵志国,唐凤鸾,等,2017.龙牙百合的研究进展[J].贵州农业科学,45(7):78-81.]