CN116286868B - 一种龙眼果实品质调控基因DlEXPA6-like及其蛋白与应用 - Google Patents
一种龙眼果实品质调控基因DlEXPA6-like及其蛋白与应用 Download PDFInfo
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Abstract
本发明提供一种龙眼果实品质调控基因DlEXPA6‑like，其核苷酸序列如SEQ ID No.1所示。本发明对DlEXPA6‑like基因进行克隆，分析该基因的序列结构、进化关系、组织表达等。同时构建过表达载体，并转化到Mico Tom番茄中进行功能分析。结果表明DlEXPA6‑like基因负调控果实发育，正调控果实数目和可溶性固形物含量。该结果不仅会为龙眼等果树果实重量/大小和品质调控理论研究的开展奠定重要基础，同时也为后续利用分子辅助育种开展大果型优质龙眼新品种选育提供重要基因资源和分子标记。
Description
技术领域
本发明涉及分子生物技术领域，具体涉及一种龙眼果实品质调控基因DlEXPA6-like基因及其应用。
背景技术
龙眼新品种到目前为止，还没有选育出能与泰国龙眼竞争具有优势的商业栽培品种。深入挖掘参与调控龙眼果实品质性状的候选基因，对于加快培育大果型优质龙眼新品种和我国龙眼产业的“提质增效”具有重要意义。Expansin蛋白的分子量一般在25-30kDa，包含250-300个氨基酸。Expansins蛋白一般含有2个保守结构域(domainΙ和domainΠ)。DomainΙ是由120-135个氨基酸残基组成的结构域，该结构域具有一个典型的HFD(His-Phe-Asp)基序，是expansins蛋白的结构特征。Expansins蛋白的N段都有一段与domainΙ相连的信号肽区，可以引导expansins蛋白定位于细胞壁。DomainΙ和domainΠ之间有一段起连接作用的氨基酸序列。DomainΠ由90-120个氨基酸残基组成，有保守的色氨酸残基，与某些细菌的纤维素结合结构有很高的相似度(Sampedro J,Cosgrove D J.The expansinsuperfamily.Genome biology,2005,6(12):1-11.)。根据氨基酸序列特征可将植物的Expansin划分为4个亚家族：α-expansin(EXPA)，β-expansin(EXPB)，expansin-like A(EXLA)和expansin-like B(EXLB)。其中，EXPA和EXPB是最大的2个亚家族(Cosgrove DJ.Plant expansins:diversity and interactions with plant cell walls.Currentopinion in plant biology,2015,25:162-172.)。Expansin蛋白可以诱导植物细胞壁发生不可逆的伸展，同时通过促进细胞壁疏松来适应植物器官发育及应对各种压力。研究表明Expansin蛋白参与植物生长的整个生命周期，包括营养生长(根、茎、叶等器官的生长)和生殖生长(花、果实、种子等生殖器官的生长)以及非生物逆境胁迫响应、器官脱落、气孔运动等各个方面(Cosgrove D J.Plant expansins:diversity and interactions with plantcell walls.Current opinion in plant biology,2015,25:162-172.；Liu B,Zhang B,Yang Z,Y Liu,S Yang,Y Shi,Jiang C,Qin F.Manipulating ZmEXPA4 expressionameliorates the drought-induced prolonged anthesis and silking interval inmaize.The Plant Cell,2021,33(6):2058-2071.；Sun Q,Li Y,Gong D,Hu A,Zhong W,Zhao H,Ning Q,Tan Z,Liang K,Mu L,Jackson D,Zhang Z,Yang F,Qiu F.A NAC-EXPANSIN module enhances maize kernel size by controlling nucelluselimination.Nature Communications,2022,13(1):1-14.)。作为最早鉴定出的亚家族，EXPA主要参与调解酸诱导型细胞壁松弛、植物根毛发育、逆境胁迫响应、果实成熟、作物产量等(Cosgrove D J.Plant expansins:diversity and interactions with plant cellwalls.Current opinion in plant biology,2015,25:162-172.；Ramakrishna P,DuarteP R,Rance G A,Schubert M,Vordermaier V,Vu LD,Murphy E,Barro AV,Swarup K,Moirangthem K,B,Cotte B,Goh T,Lin Z,VoβU,Beeckman T,Bennett M,GevaertK,Maizel A,Smet V.EXPANSIN A1-mediated radial swelling of pericycle cellspositions anticlinal cell divisions during lateral rootinitiation.Proceedings of the National Academy of Sciences,2019,116(17):8597-8602.；Yang J,Zhang G,An J,Q Li,Y Chen,X Zhao,J Wu,Y Wang,Q Hao,W Wang,WWang.Expansin gene TaEXPA2 positively regulates drought tolerance intransgenic wheat(Triticum aestivum L.).Plant Science,2020,298:110596.；Dong C,Zou X,Gao Q.Genome-wide identification of expansin in Fragaria vesca andexpression profiling analysis of the FvEXPs in different fruitdevelopment.Gene,2022,814:146162.；Wu Z,Li M,Zhong Y,Li L,Cheng D,Gu H,Guo X,Qi X,Chen J.Overexpression of AcEXPA23 promotes lateral root development inkiwifruit.International Journal of Molecular Sciences,2022,23(14):8026.)。植物细胞壁在植物发育过程中起到重要作用，同时也限制植物细胞原生质的增加，因此，植物细胞壁是限制细胞增大的主要因素(Taiz L.Expansins:proteins that promote cell wallloosening in plants.Proceedings of the National Academy ofSciences of theUnited States of America,1994,91(16):7387.)。Expansin蛋白主要破坏细胞壁纤维素和半纤维素之间的非共价键，细胞壁受到来自原生质体膨胀压力，从而使得纤维素与半纤维素之间有一个短暂的滑动过程，而且是不可逆的。目前，关于expansin蛋白参与器官发育调控和影响产量的研究都聚焦于模式植物和大宗农作物中。过表达OsEXP4，12％的转基因水稻植株高于对照植株，而88％的转基因植株要比对照矮，同时多数转基因植株都至少多发育出2个叶片，胚芽鞘和中胚轴长度也分别增加了31％和97％，而基因沉默株系出现了相反的表型。分析表明植株不同器官大小的改变与OsEXP4的表达水平显著正相关。解剖学分析表明OsEXP4可能是通过调节对应器官的细胞壁扩张，从而影响细胞和器官的大小(ChoiD,Lee Y,Cho H T,Kende H.Regulation of expansin gene expression affects growthand development in transgenic rice plants.The Plant Cell,2003,15(6):1386-1398.)。然而，对于expansin蛋白与果实器官发育关系还鲜有报道。
发明内容
本发明目的在于提供一种龙眼果实品质调控基因。
本发明另一目的在于提供上述龙眼果实品质调控基因表达的蛋白。
本发明又一目的在于提供上述龙眼果实品质调控基因的应用。
本发明目的按如下技术方案实现：
一种龙眼果实品质调控基因DlEXPA6-like，其核苷酸序列如SEQ ID No.1所示。
一种龙眼果实品质调控蛋白，其氨基酸序列如SEQ ID No.2所示。
本发明还提供了含有前述编码基因的载体。
本发明还提供了含有前述载体的工程菌。
本发明进一步提供了前述基因在龙眼果实大小、数目和品质调控方面的应用。
进一步地，所述应用为将所述工程菌侵染植株，获得果实大小、数目和品质调控的转基因植株。
本发明具有如下有益效果：
本发明以DlEXPA6-like基因为对象，克隆了该基因的ORF全长，分析该基因的序列结构、进化关系、组织表达情况等。qRT-PCR分析表明该基因在F1代中大果株系FD105和小果株系FD21的不同发育阶段果实表现出差异表达。
本发明对DlEXPA6-like基因进行克隆，分析该基因的序列结构、进化关系、组织表达等。同时构建过表达载体，并转化到Mico Tom番茄中进行功能分析。结果表明，DlEXPA6-like基因含有expansin蛋白保守的domainΙ和domainΠ结构域，说明DlEXPA6-like是典型扩张蛋白，与来自阿月浑子等果树的EXPA6-like亚家族成员关系更近，具有组织表达特异性，在叶片中的表达量最低，在花和幼果中表达量较高；在F1代的小果株系FD21果实发育的60-70DAP和80～90dDAP内显著下调表达，在大果株系FD105果实发育的70～80DAP内显著下调表达，过表达转基因株系的果实也明显小于野生型Mico Tom番茄的果实，果实数目显著增多，果实的可溶性固形物含量显著上调。以上结果表明DlEXPA6-like基因负调控果实大小和重量，正调控果实数目和可溶性固形物含量。该结果不仅会为龙眼等果树果实重量/大小和品质调控理论研究的开展奠定重要基础，同时也为后续利用分子辅助育种开展大果型优质龙眼新品种选育提供重要基因资源和分子标记。
附图说明
图1：DlEXPA6-like基因PCR扩增图。
图2：DlEXPA6-like与龙眼(“红核子”)基因组EXPA6-like(Dlo_024180.1)的碱基和氨基酸序列对比。红色箭头表示碱基或氨基酸不同处。
图3：不同物种间expansin蛋白序列比对图。框部分表示expansin蛋白保守的domainΙ和domainΠ结构域氨基酸序列。
图4：龙眼DlEXPA6-like与GenBank中相似序列的进化树分析图。
图5：DlEXPA6-like在龙眼不同组织中的相对表达量图。不同字母靶标表示差异达到显著水平。
图6：DlEXPA6-like在不同F1后代果实发育中的相对表达量图。
图7：DlEXPA6-like转基因番茄果实发育表型图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1目的基因的克隆
材料和方法
1.1植物材料
选取3组在长势和树龄(9年龄)一致的‘四季蜜′龙眼为取样树，取‘四季蜜′龙眼的花、花芽、叶、果皮、果肉、根、种子、茎和幼果(花后60d整果)等器官为材料进行组织表达分析。选取3组在长势和树龄(10年龄)一致的F1代大果型株系FD105和小果型株系FD21龙眼(F1代父母本是‘凤梨朵′(母本)ב大乌圆′(父本))为取样树，取花后60、70、80、90和100d的龙眼果肉为材料进行果实发育分析。采集FD21龙眼叶片作为基因克隆模板材料。所有的试验设3次重复，取样后立即放入液氮速冻并转入-80℃冰箱中保存、备用。
1.2DlEXPA6-like基因序列的克隆及生物信息分析
从龙眼基因组数据库(NCBI Sequence Read Archive，SRA315202)中获得DlEXPA6-like基因(Dlo_002630.2)的碱基序列和氨基酸序列信息。利用PrimerPremier5.0根据DlEXPA6-like基因的ORF序列设计引物EXPA6-like-S和EXPA6-like-A(表1)，委托天一辉远生物科技有限公司(广州)合成。用北京华越洋生物公司的植物RNA提取试剂盒提取FD21龙眼叶片的RNA，采用Takara公司的PrimeScript RT-PCR试剂盒，具体操作步骤参照说明书，反转录cDNA作为模板，进行PCR扩增克隆DlEXPA6-like基因。扩增条件为：94℃预发性5min；94℃发性30s，60℃退火30s，72℃延伸60s，35个循环(发性-延伸)；72℃延伸10min，4℃保存。扩增产物进行切胶回收和纯化连接到pMD18-T载体上，转化DH5α感受态细胞，PCR筛选阳性克隆，挑取阳性单克隆送天一辉远生物科技有限公司(广州)进行测序。
利用在线软件SMART程序(http://smart.emblheidelberg.de/)预测蛋白结构域，利用ExPASy(http://expasy.org/tools/)分析蛋白的等电点和分子量。根据克隆所得的cDNA序列，利用BLASTp对氨基酸序列进行同源性对比，同时利用MEGA 5软件进行氨基酸序列同源性分析及系统发育分析，构建Neighbor-Joining进化树，1000次重复，其它均为默认设置。
1.3表达分析
根据克隆所得的DlEXPA6-like基因序列设计qRT-PCR引物qEXPA6-like-S和qEXPA6-like-A(表1)，并在NCBI里利用BLASTn检验以确保引物的特异性。以龙眼的Actin基因(Dlo_028674)为内参基因，具体引物序列见表1。
表1所用引物信息
Tab.1 Information of primers used
qRT-PCR反应所用仪器为Roche的LightCycler 480，PCR反应酶为Takara公司的SYBR Green Master Mix。反应体系为20mL，其中模板cDNA 40ng，上、下游引物各250nM，SYBR Green Master Mix 10μL，其余用ddH2O补齐。反应程序：94℃预发性5min；94℃10s，59℃20s，72℃30s，40个循环后作熔解曲线(95→65℃，0.1℃/s)。利用2-ΔΔCt计算DlEXP-likeA基因的相对表达量。所有样品进行3次重复，均设阴性对照。采用Excel软件进行平均数统计，以SPSS软件进行单因素方差分析目的基因在不同组织和材料里的发化的差异显著性(P<0.05)，并使用SigmaPlot 12.5软件作图。
实施例3过表达载体构建及转基因番茄功能验证
使用特异PCR引物OEEXPA6-like-S/OEEXPA6-like-A(表1)，以龙眼cDNA为模板，进行PCR扩增。该引物5′端分别加有BamH I酶切位点，反引物5’端分别加有Sac I酶切位点。获得的PCR产物与pMD19-T载体连接并进行测序。最后，提取测序正确的质粒，用BamH I和SacI分别双酶切pBI121和测序正确的质粒，通过T4 DNA连接酶构建含有DlEXPA6-like目标基因的植物表达载体，并命名为pBI121-DlEXPA6-like。通过液氮冻融法将所构建的过量表达载体pBI121-DlEXPA6-like转入农杆菌菌株GV3101，参照文献(Arshad W,Waheed M T,Mysore K S,et al.Agrobacterium-mediated transformation of tomato with rolBgene results in enhancement of fruit quality and foliar resistance againstfungal pathogens[J].PLoS One,2014,9(5):e96979.)，通过农杆菌侵染法将DlEXPA6-like基因转入番茄(Micro-Tom)，获得T0代种子。在含30ug/ml的MS固体培养基上筛选阳性番茄，同时用pBI121质粒特异引物检测阳性转基因番茄幼苗。将T3代转基因植株分别和野生型在相同环境中培养，并比较它们的果实发育表型。
实施例4结果与分析
1.DlEXPA6-like基因的克隆及生物信息学分析
以FD21龙眼叶片cDNA为模板，用EXPA6-like-S/EXPA6-like-A(表1)引物扩增出800bp左右的片段(图1)。测序结果显示。该基因大小为786bp，编码261个氨基酸，其分子量为28.16kDa，理论等电点为9.50。我们获得的DlEXPA6-like基因与已知龙眼(“红核子”)基因组数据库(NCBI Sequence Read Archive，SRA315202)中EXPA6-like基因(Dlo_002630.2)相比，在碱基序列中存在3处不同(44处的T-G；67-68处的GC-AG)，导致氨基酸序列存在两处不同(15处的F-C；23处的A-S)(图2)。根据与其他作物expansin家族成员的亲缘关系，命名为DlEXPA6-like。氨基酸序列分析表明DlEXP-like A与阿月浑子PVEXPA6(Pistacia vera,XP_031273596.1)；栎树QrEXPA4(Quercus robur,XP_050290897.1)和杧果MiEXPA6(Mangifera indica,XP_044482542.1)一样，含有expansin蛋白保守的domainΙ和domainΠ结构域，说明DlEXPA6-like是典型扩张蛋白(图3)。
利用BLASTp对DlEXP-like A的氨基酸序列进行同源性检索，然后利用MEGA 6.0软件构建系统进化树(图4)。结果表明，DlEXP-like A在进化上与玉米的ZmEXPA4、水稻的OsEXP4拟南芥的AtEXPA4和AtEXPA16的亲缘较近，被划分在EXP-like A亚家族里。
3、DlEXPA6-like基因组织表达特性分析
qRT-PCR结果表明DlEXPA6-like基因在被检测的9种龙眼组织中都有表达，但表达具有组织特异性，其中在叶片中表达最低，在根中的表达量最高(约是叶片中表达的998倍)。在花和幼果中的表达量都较高(图5)。该结果说明DlEXPA6-like基因可特异参与龙眼根、花和幼果的发育。
4、DlEXPA6-like基因在花、果发育过程中的表达模式
利用qRT-PCR技术，我们分析了DlEXPA6-like在F1代大果型株系FD105和小果型株系FD21果实发育过程中的表达模式。结果显示，在果实发育早期60～70d以及80～90d，DlEXPA6-like基因在FD21中呈显著下调趋势。在果实发育后期70～80d，DlEXPA6-like基因在FD105中呈下调趋势(图6)。该结果表明，DlEXP-like A基因表达与果肉器官发育有相关性。
5、转DlEXPA6-like基因的番茄表型分析
转基因表型结果显示，过表达DlEXPA6-like基因的番茄植株的果实相对于野生型显著发小(降低37.83％)，横经和纵经也显著降低(分别降低32.95％和35.66％)。但同时，果实数目显著增多近1.26倍，可溶性固形物含量提升55.87％(图7)，该结果说明DlEXPA6-like基因过量表达会显著降低果实大小和重量，同时增加果实数目和可溶性固形物含量，可以作为大果优质龙眼新品种选育候选基因。
Claims (2)
1.一种龙眼果实品质调控基因DlEXPA6-like在过表达负调控龙眼果实大小、正调控龙眼果实数目和可溶性固形物含量的应用，所述调控基因DlEXPA6-like核苷酸序列如SEQ IDNo.1所示。
2.一种龙眼果实品质调控基因DlEXPA6-like表达的调控蛋白在过表达负调控龙眼果实大小、正调控龙眼果实数目和可溶性固形物含量的应用，所述调控蛋白的氨基酸序列如SEQ ID No.2所示。
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