CN109207493B - 一种梨漆酶基因PbLAC1及其载体、宿主细胞和应用 - Google Patents
一种梨漆酶基因PbLAC1及其载体、宿主细胞和应用 Download PDFInfo
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- CN109207493B CN109207493B CN201811170973.4A CN201811170973A CN109207493B CN 109207493 B CN109207493 B CN 109207493B CN 201811170973 A CN201811170973 A CN 201811170973A CN 109207493 B CN109207493 B CN 109207493B
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Abstract
本发明涉及植物分子生物学和基因工程技术领域,提供了一种梨漆酶基因PbLAC1,核苷酸序列如SEQ ID NO.1所示。本发明还提供了一种植物超量表达载体,所述植物超量表达载体包括PbLAC1构建的pMD‑18T‑PbLAC1质粒。本发明还提供了一种遗传工程化的宿主细胞,所述宿主细胞包括PbLAC1基因序列。本发明还提供了上述梨漆酶基因PbLAC1在调控植物木质素合成及细胞壁发育中的应用。本发明的梨漆酶基因PbLAC1为完善植物木质素合成途径提供了新的证据,对水果品质改良以及作物其他关键农艺性状的改良也具有重要的理论及实践意义,同时,还为利用基因工程调控梨石细胞含量提供了新的途径。
Description
技术领域
本发明涉及植物分子生物学和基因工程技术领域,尤其涉及一种梨漆酶基因PbLAC1及其载体、宿主细胞和应用。
背景技术
砀山酥梨(Pyrus bretshneideri cv.DangshanSu),属白梨(Pyrusbretshneideri)系梨品种,原产地为安徽省砀山县,现为我国华北、西北、黄河流域等地主要的栽培品种。砀山酥梨有着悠久的栽培历史,声誉在外,战国时期庄子就在《人世间》中记载:夫揸梨橘柚,果蔬之属,实熟则剥,剥则辱,大枝折,小枝泄。这是可考的对砀山酥梨的最早的文字描述。清朝高宗皇帝曾出宫南巡,途径徐州食砀山酥梨后,对其称赞有加,称:进贡梨果甚多,此梨实甲天下也。并召砀山酥梨回京呈供皇考祭祀。然而,近年来,随着大量新的、品质更好的梨品种进入了消费者的视野,砀山酥梨与其他梨品种相比,便逐渐表现出石细胞含量较高、果实品质较差的特点,并最终影响到其市场份额。因此,降低石细胞含量,进一步提高果实品质逐渐成为如今砀山酥梨科研工作者研究的重要目标。
梨石细胞在分类上属短石细胞,在梨果实中单个或成群存在,成群的石细胞称为石细胞团或石细胞簇。石细胞团的大小、数目和密度能直接影响梨果实品质。梨石细胞团直径在0.15mm以下时不影响口感,通常在食用时不易察觉;而石细胞团大小在0.20mm以下时,肉质细,口感好;若石细胞团直径在0.30mm以上,则能明显感到果肉质地粗,口感多渣。此外,梨果实中糖分与石细胞含量呈现显著的负相关。
目前大量研究发现,砀山酥梨果实石细胞的形成是从薄壁细胞细胞壁不均匀木质化开始的,在整个发育时期,果实木质素含量与石细胞含量呈显着正相关,发育成熟的梨石细胞中含有约40%的木质素。石细胞整个木质化过程中,其细胞壁按照木质化程度不同可以分为复合胞间层、次生壁S1层、次生壁S2层以及次生壁S2L层等,其中,次生壁S2L层木质化程度最高。由此可见,木质素是石细胞的关键组分之一,其生物合成对于石细胞的发育至关重要。
木质素(Lignin)的基本单元是苯丙烷,是一种高聚物,具芳香族特性,非晶体。整个木质素单体的生物合成从苯丙氨酸的脱氨反应开始,第一步由苯丙氨酸氨裂解酶催化,经芳环修饰,发生羟化、甲基化、转化羧酸,部分通过酯化反应催化,最终产生三种木质素单体:香豆醇(H型木质素)、松柏醇(G型木质素)和芥子醇(S型木质素)。目前大量研究均集中在木质素单体合成相关结构基因上,对于木质素单体合成后聚合机制的研究尚且薄弱。迄今为止,木质素单体聚合分子机理尚不明确。
漆酶(EC1.10.3.2)全称为对-二酚:(双)氧化还原酶,属于铜蓝氧化酶家族成员,是一种铜离子氧化酶。早在19世纪,日本人就在漆树分泌液中发现了漆酶。1898年,漆酶(Laccase)这个概念被首次提出并沿用至今。Bertrand于1985年首次鉴定漆酶为一种金属蛋白,每个蛋白单体中含有四个铜离子,部分单体中含有其他金属离子如锰、铁、锌离子等。漆酶来源广泛,在高等植物、真菌、昆虫中都有关于漆酶的报道。植物漆酶主要与植物的抗逆、多酚类物质氧化聚合等相关,其中就包括木质素单体的聚合。
木质素单体经合成后要通过脱氢反应,产生共振稳定的自由基,才能形成木质素高聚合物,而漆酶就是催化木质素单体氧化/脱氢反应的酶之一。拟南芥漆酶家族(AtLAC)有17个成员,其中LAC4和LAC17的突变能使木质素含量降低30~40%。而AtLAC4,AtLAC11和AtLAC17同时突变可以引起严重的木质素缺陷型生长障碍。甘蔗漆酶在拟南芥AtLAC17突变体中过表达可使其木质素含量基本恢复野生型水平。过表达棉花漆酶(GaLAC1)的转基因植物表现出2.1至13.2倍的漆酶活性,且茎中的总木质素含量增加2.1~19.6%。以上实验均能证明漆酶在木质素合成中起着至关重要的作用。
然而,目前关于梨中漆酶的克隆及功能分析鲜有报道,到底梨漆酶家族哪些成员在木质素生物合成中发挥作用也不明确。因此,挖掘梨中木质素合成相关的漆酶成员,对于调控梨果实中石细胞发育、改善梨品质具有重要意义。此外,木质素的含量高低与水果(杏、石榴、葡萄等)的果核大小及硬度、农作物抗倒伏能力以及秸秆降解再利用均密切相关,因此,挖掘梨中木质素合成相关的漆酶成员,对于改良园艺作物及粮食作物关键农艺性状也具有重要意义。
发明内容
本发明所要解决的技术问题在于提供一种既能调控梨果实中石细胞发育、改善梨品质,又能进一步地改良园艺作物及粮食作物关键农艺性状的梨漆酶基因PbLAC1及其载体、宿主细胞和应用。
本发明采用以下技术方案解决上述技术问题:
一种梨漆酶基因PbLAC1,所述梨漆酶基因PbLAC1的核苷酸序列如SEQ ID NO.1所示。
作为本发明的优选方式之一,所述核苷酸序列编码的氨基酸序列如SEQ ID NO.2所示,核苷酸长度为1755bp,氨基酸长度为585个氨基酸。
作为本发明的优选方式之一,所述梨漆酶基因PbLAC1的克隆步骤如下:提取梨果实总RNA并反转录成cDNA,根据公布的梨基因组序列设计特异性扩增引物PbLAC1-F和PbLAC1-R,以cDNA为模板进行PCR扩增;其中,特异性扩增引物PbLAC1-F和PbLAC1-R的核苷酸序列分别如SEQ ID NO.3、SEQ ID NO.4所示。
一种上述梨漆酶基因PbLAC1在调控植物木质素合成及细胞壁发育中的应用。
一种植物超量表达载体,所述植物超量表达载体包括上述梨漆酶基因PbLAC1构建的pMD-18T-PbLAC1质粒。
作为本发明的优选方式之一,所述植物超量表达载体采用pMD-18T-PbLAC1质粒和pCambia1304质粒进行双酶切构建植物超量表达载体pCambia1304-PbLAC1。
一种遗传工程化的宿主细胞,所述宿主细胞包括上述梨漆酶基因PbLAC1基因序列。
作为本发明的优选方式之一,将所述梨漆酶基因PbLAC1构建的植物超量表达载体pCambia1304-PbLAC1质粒转化至感受态细胞中,即得。
作为本发明的优选方式之一,所述植物超量表达载体pCambia1304-PbLAC1质粒采用pMD-18T-PbLAC1质粒和pCambia1304质粒进行双酶切构建而成。
作为本发明的优选方式之一,所述宿主细胞具体采用根癌农杆菌(Agrobacterium)EHA105(C58(rif R)Ti pEHA105(pTiBo542DT-DNA)(strepR)Succinamopine)。
作为本发明的优选方式之一,所述根癌农杆菌(Agrobacterium)EHA105(C58(rifR)Ti pEHA105(pTiBo542DT-DNA)(strepR)Succinamopine)直接购买于现有的国内外市场。
梨漆酶基因PbLAC1在调节植物木质素合成及细胞壁发育中的作用机理:梨漆酶基因PbLAC1对植物木质素合成及细胞壁发育起正调控作用,其超量表达可提升植株中木质素含量及细胞壁厚度;同理,在梨果实中抑制该基因表达则会降低木质素含量及细胞壁厚度,从而抑制石细胞发育,提升果实品质。
本发明相比现有技术的优点在于:
梨漆酶基因PbLAC1可以直接促进植物木质素的生物合成和细胞壁发育,提高木质化程度;在植物中超量表达该基因可以提高植物茎干的刚性,增强抗倒伏能力;在梨果实中抑制该基因表达可抑制石细胞发育,提升果实品质;而且,梨果实表面的果锈也与木质素合成相关,抑制该基因可以同时提高梨的内在及外观品质。据此,梨漆酶基因PbLAC1为完善植物木质素合成途径提供新的证据,对水果品质改良(石细胞含量、果核大小及硬度等)以及作物其他关键农艺性状(如抗倒伏等)的改良也具有重要的理论及实践意义,同时,还为利用基因工程调控梨石细胞含量提供了新的途径。
附图说明
图1是实施例2中梨漆酶基因PbLAC1超量表达载体pCambia1304-PbLAC1的构建图谱;
图2是实施例3中梨漆酶基因PbLAC1超量表达拟南芥T3代植株GUS染色图;
图3是实施例3中野生型拟南芥与梨漆酶基因PbLAC1超量表达拟南芥花序茎的甲苯胺蓝染色结果对比图(图中,A为野生型拟南芥的甲苯胺蓝染色结果对比图,B为超量表达PbLAC1的T3代拟南芥的甲苯胺蓝染色结果图);
图4是实施例3中野生型拟南芥与梨漆酶基因PbLAC1超量表达拟南芥花序茎的Wiesner染色结果对比图(图4中,A为野生型拟南芥的Wiesner染色结果图,B为超量表达PbLAC1的T3代拟南芥的Wiesner染色结果图);
图5是实施例3中野生型拟南芥与梨漆酶基因PbLAC1超量表达拟南芥花序茎木质素含量检测结果图;
图6是实施例3中梨果实不同发育时期PbLAC1表达量分析柱状图(图中,横坐标表示果实发育时期,纵坐标表示PbLAC1表达量);
图7是实施例3中梨不同组织部位PbLAC1表达量分析柱状图(图中,横坐标由左至右依次表示为Fruit、Bud、Stem、Flower、Leaf,纵坐标表示PbLAC1表达量);
图8是实施例3中梨漆酶基因PbLAC1氨基酸序列系统发育树聚类分析图。
具体实施方式
下面对本发明的实施例作详细说明,本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
本发明的一种梨漆酶基因PbLAC1及其载体、宿主细胞和应用,下述实施例中所用方法如无特别说明均为常规方法,所用引物均由上海生工生物工程技术服务有限公司合成,测序由上海生工生物工程技术服务有限公司进行,pMD-18T、限制性内切酶Nco I和BglII、T4连接酶均购自宝日医生物技术(北京)有限公司(Takara中国)。RNAprep Pure PlantKit、FastQuant RT Kit均购自TIANGEN公司。DNA胶回收试剂盒、质粒提取试剂盒购、X-Gluc均购自上海生工生物工程技术服务有限公司,方法均照说明书进行。梨为砀山酥梨。
实施例1
本实施例的一种梨漆酶基因PbLAC1,克隆步骤为:选取砀山酥梨花后39天左右的果实,提取总RNA并反转录成cDNA,再根据公布的梨基因组序列设计特异性扩增引物PbLAC1-F和PbLAC1-R,并以cDNA为模板进行PCR扩增,接着进行PCR产物的回收以及连接pMD-18T载体;其中,特异性扩增引物PbLAC1-F和PbLAC1-R的核苷酸序列分别如SEQ IDNO.3、SEQ ID NO.4所示。
具体克隆过程如下:
(1)取1μg(2μL)的总RNA,加入2μL 5×gDNA Buffer,2μL 10×Fast RT Buffer,1μL RT Enzyme Mix,2μL FQ-RT Primer Mix,11μL RNase-Free ddH2O,小心混匀,42℃孵育15min,然后95℃孵育3min,冰上放置5min,即获得相应的反转录产物cDNA。
(2)以反转录合成的cDNA为模板,利用引物PbLAC1-F和PbLAC1-R进行PCR扩增;反应程序为:94℃5min;94℃30s,48℃30s,72℃45s,35个循环;72℃10min。
(3)PCR产物经1%琼脂糖凝胶电泳检测后,对目的片段进行回收,并连接到载体pMD-18T上,然后转化到大肠杆菌DH5α感受态细胞,阳性克隆鉴定后委托上海生工生物工程技术服务有限公司进行测序。
测序完成后,利用DNAMAN软件进行比对,确保所获序列为目的序列。结果表明:所获基因大小为1755bp,其核苷酸序列如SEQ ID NO.1所示;核苷酸序列编码的氨基酸序列如SEQ ID NO.2所示,氨基酸长度为585个氨基酸。
实施例2
本实施例的一种PbLAC1超量表达载体及宿主细胞的构建:
(1)根据已经成功克隆的目的基因PbLAC1序列设计带有Bgl II和Nco I酶切位点的引物PbLAC1-ZH-F和PbLAC1-ZH-R,以已构建的pMD-18T-PbLAC1质粒为模板进行扩增,PCR产物经1%琼脂糖凝胶电泳检测后,对目的片段进行回收,并连接到载体pMD-18T上进行测序;其中,带有Nco I和Bgl II酶切位点的引物PbLAC1-ZH-F和PbLAC1-ZH-R的核苷酸序列分别如SEQ ID NO.5、SEQ ID NO.6所示。
(2)用限制性内切酶Bgl II和Nco I分别对pMD-18T-PbLAC1质粒和植物表达载体pCambia1304质粒进行双酶切;酶切产物用1%的琼脂糖凝胶电泳,回收目的片段PbLAC1和植物表达载体pCambia1304;用T4连接酶将2个酶切后的目的片段连接并转化到大肠杆菌DH5α感受态细胞,重组质粒进行酶切验证后并进行测序验证,最终获得植物超量表达载体pCambia1304-PbLAC1(图谱如图1所示)。
(3)将构建好的植物表达载体pCambia1304-PbLAC1通过电转化到农杆菌EHA105感受态细胞中,并通过卡那霉素和利福平筛选出阳性菌株,获得宿主细胞。
实施例3
本实施例的一种PbLAC1超表达拟南芥株系的构建:
(1)制备侵染液:
将实施例2中获得的宿主细胞活化后,用缓冲液重悬,获得侵染液;其中,缓冲液具体为含有质量比为5%的蔗糖和0.02%的Silwet L-77的1/2MS液体培养基,侵染液的OD600值为0.5~0.8。
(2)拟南芥的遗传转化:
利用农杆菌介导的浸花法完成。
(3)转基因植株筛选:
将T0代种子消毒后接种至含有50mg/L潮霉素的MS固体筛选培养基中,置于光照培养箱中培养,并控制光照时间为16h/d;结果显示:非转基因植株黄化并停止生长,只有转基因植株可以生长。
(4)阳性转基因植株鉴定:
取少部分叶片,利用EasyPure Plant Genomic DNA Kit提取拟南芥叶片的DNA,PCR鉴定时,利用pCambia1304载体上的GFP基因的引物检测;反应体系为:DNA 2μL,Easytaqbuffer 2.5μL,dNTPs 2.0μL,上下游引物各1.5μL,Easytaq 0.25μL,水补齐至25μL;反应条件为:94℃5min,94℃30s,55℃30s,72℃45s,30个循环,72℃10min。其中,设计的引物具体为GFP-F、GFP-R,GFP-F的序列如SEQ ID NO.7所示,GFP-R的序列如SEQ ID NO.8所示。
待转基因阳性株长出4-6片莲座叶后,移入营养土中继续培养并收获T1代种子。
(5)将T1代种子重复步骤进行复筛,获得遗传稳定的T3代拟南芥阳性株。
(6)转基因拟南芥植株的GUS染色:
取拟南芥阳性株T3代植株的幼苗作为实验对象,按照GUS染色试剂盒对其进行GUS染色,染色结果如图2所示;分析图2可知,转化pCambia1304-PbLAC1的拟南芥幼苗的根、茎、叶均出现蓝色,由此表明PbLAC1在转基因拟南芥的各部位都成功表达。
(7)拟南芥花序茎组织化学染色和显微观察:
取超量表达PbLAC1的T3代拟南芥与野生型拟南芥同位置的花序茎段,徒手切片,将切片分别进行甲苯胺蓝染色和Wiesner染色。
甲苯胺蓝染色:利用1%甲苯胺蓝浸泡后直接封片观察,甲苯胺蓝可以将植物细胞壁特异性染成蓝色。
Wiesner染色:切片用2%间苯三酚(溶于95%乙醇)染色5min,转入15%(v/v)HCl浸泡3min后直接封片观察。
结果分析:甲苯胺蓝染色结果如图3所示(图3中,A图为野生型拟南芥的甲苯胺蓝染色结果图,B图为超量表达PbLAC1的T3代拟南芥的甲苯胺蓝染色结果图),Wiesner染色结果如图4所示(图4中,A图为野生型拟南芥的Wiesner染色结果图,B图为超量表达PbLAC1的T3代拟南芥的Wiesner染色结果图)。由图3可知,与野生型拟南芥相比,PbLAC1超量表达植株花序茎中木质部和束间纤维细胞的细胞壁显著增厚。由图4可知,PbLAC1超量表达植株的染色区域与野生型拟南芥相比增加,且着色程度较深(Wiesner染色可以将木质素特异性染成紫红色),这说明PbLAC1超量表达植株的木质部和束间纤维区域的木质化程度升高。
(8)转基因拟南芥花序茎木质素含量检测:
利用紫外分光光度法(溴乙酰-冰乙酸),木质素的含量按下列公式计算:
式中,吸光标准为拟南芥木质素标准吸光率,为17.2。
野生型拟南芥与超量表达PbLAC1拟南芥花序茎的木质素含量测定均有三次生物学重复。显著性分析利用使用Statistical Program for Social Sciences软件(版本19.0,IBM,www.ibm.com)和Microsoft Excel 2010完成。
结果分析:图5为野生型拟南芥与PbLAC1超量表达拟南芥花序茎木质素的含量检测结果图(WT表示野生型)。由图5可知,野生型拟南芥中木质素含量为12.67%,超量表达PbLAC1拟南芥中木质素含量分别为14.90%、14.80%。由此可见,超量表达PbLAC1拟南芥中木质素含量均显著高于野生型拟南芥,约增加了17%。
(9)PbLAC1基因的时空表达模式分析及系统进化分析:
根据PbLAC1基因的核苷酸序列SEQ ID NO.1信息及梨基因组信息,利用PrimerPremier 5.0软件设计其荧光定量引物PbLAC1-Q-F(序列见SEQ ID NO.9)和PbLAC1-Q-R(序列见SEQ ID NO.10)。
表1为荧光定量PCR反应体系组成表,按照表1将各组分加入八连管中,以梨Tubulin(Accession No.AB239680.1)作为内参基因,每个样品进行三次生物学重复。以2-△△CT法计算各基因的相对表达水平。PCR反应程序为:98℃2min,98℃变性10s,60℃退火10s,68℃延伸30s,40个循环;后做65℃到99℃溶解曲线分析。所用仪器为GFX96Real-Time荧光定量PCR仪。
表1荧光定量PCR反应体系
结果分析:图6为梨果实不同发育时期(不同发育天数)PbLAC1表达量分析柱状图(图中,横坐标表示果实发育时期,纵坐标表示PbLAC1表达量),图7为梨不同组织部位PbLAC1表达量分析柱状图(图中,横坐标由左至右依次表示为Fruit(果实)、Bud(芽)、Stem(茎)、Flower(花)、Leaf(叶),纵坐标表示PbLAC1表达量)。由图6、图7可知,PbLAC1的表达趋势总体上与木质素含量、石细胞含量呈正相关。在木质素和石细胞大量积累的时期表达水平较高,在果实发育后期表达量下降。此外,PbLAC1主要在果实和茎中表达。
利用MEGA 5.1软件采用邻接法(Neighbor-joining)漆酶构建种间系统发育树,系统发育树聚类表明(PbLAC1氨基酸序列系统发育树聚类分析结果如图8所示),PbLAC1与已知其他物种中木质素合成相关漆酶亲缘关系较近,如AtLAC17、SofLAC、BdLAC5等。
综合上述各实施例可知,梨漆酶基因PbLAC1具有合成植物木质素、促进细胞壁增厚的作用,在植物体内超量表达该基因可导致植物体中木质素含量上升且细胞壁增厚;据此,该基因在调节植物木质素合成及细胞壁发育方面表现出了优越的应用潜能。
本实施例梨漆酶基因PbLAC1的具体优势如下:
梨漆酶基因PbLAC1可以直接促进植物木质素的生物合成和细胞壁发育,提高木质化程度;在植物中超量表达该基因可以提高植物茎干的刚性,增强抗倒伏能力;在梨果实中抑制该基因表达可抑制石细胞发育,提升果实品质;而且,梨果实表面的果锈也与木质素合成相关,抑制该基因可以同时提高梨的内在及外观品质。据此,梨漆酶基因PbLAC1为完善植物木质素合成途径提供新的证据,对水果品质改良(石细胞含量、果核大小及硬度等)以及作物其他关键农艺性状(如抗倒伏等)的改良也具有重要的理论及实践意义,同时,还为利用基因工程调控梨石细胞含量提供了新的途径。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 安徽农业大学
<120> 一种梨漆酶基因PbLAC1及其载体、宿主细胞和应用
<130> 2018
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 1755
<212> DNA
<213> 人工序列
<400> 1
atggcgtctt ctattccctt ttcaccaaca tttctagtgg cttttcttct tgttcttatc 60
tctctcttgg cctctcctga gtttgccatt gctgcagctg gcagtgttac aaggcattac 120
aaattcgata taaagttgaa aaatgtgact aggctgtgca ccacaaagag cattgtgaca 180
gtgaatggca agttccctgg acctcgaatt attgccagag aaggagacag ggttttggtt 240
aacgtggtca atcatgttcc aaacaacatg accatccatt ggcatggcat tagacaattg 300
cagaatggat gggcagatgg gccatcatat atcactcagt gccctattca aacaaaccag 360
agttatgtgt acaacttcac catcattggc caaaggggaa ctctcttctg gcatgctcac 420
atctcatggc taagagctac tgtctatgga cccctcatca tcctccctaa gcgccatgct 480
ccgtaccctt tccccaaacc ccacaaagaa gttcccatca tttttggtga gtggtggaat 540
gttgacacag aggctgtaat tagccaggct ttacaaaccg gaaatggtcc aaatgtctcc 600
aatgcctata cgattaacgg acttccaggg ccattgtaca attgttccaa aaaggacact 660
ttcagtctaa aggtgaagcc tggaaagaca tacctcttgc gattgatcaa cgctgcagtc 720
aatgatgaac tgtttttcag catagcaaac cacagcctaa cagtagttga agcagatgct 780
gtgtatgtca aacccttcac gaccaatatt ttattcatta caccagggca aaccacaaac 840
gtgcttctca aggcaaaacc tcaataccct aatgccactt tcctcatgtt agccagaccc 900
tactttacag gcaggggtac ttttgacaat tcaactgttg ctggaattct cgagtacaaa 960
gaacactcaa attcttcttc ctccacatca ctgaaaagcc cactttttag gccaatcctt 1020
ccccaaatca atgctacttc ttttgttgca aattttacta gtaaattcag gagtttggcc 1080
agttctaaat ttccagccaa tgtgcccaga actgtcgaaa aacgcttctt tttcacagta 1140
gggcttggaa ccaacccatg ccctaaaaac caaacatgcc aagggcctac caacaccacc 1200
aaatttgcag cttctattaa caacaactca tttgctcttc cttcggtagc gctgcttcaa 1260
tctcatttct ttgctcaatc caacagtgtt tacaccaccg atttccctac tattcctccc 1320
aagccgttca attatacagg cactccacct aacaacacca atgtgagtac tgggacaaag 1380
gtggtggtgt taaagttcaa tactagtgtg gaattggtgt tgcagggcac cagcattttg 1440
ggtgctgaga gtcaccctct tcaccttcat ggcttcaact tttttgttgt tggacaaggt 1500
tttggaaact ttgaccctaa caaagacccg cctaaattca accttgtgga tcctgttgaa 1560
aggaacacct acggagttcc agccaggggt tgggcggcta tccgatttct cgcagataac 1620
ccaggcgttt ggctcatgca ttgccacttt gatgtccatt tgagctgggg gctgaggatg 1680
gcttgggtag tccaagatgg gaagcttcct aatcaaaagc tgcctcctcc accgtccgat 1740
ctcccaaagt gttga 1755
<210> 2
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Met Ala Ser Ser Ile Pro Phe Ser Pro Thr Phe Leu Val Ala Phe Leu
1 5 10 15
Leu Val Leu Ile Ser Leu Leu Ala Ser Pro Glu Phe Ala Ile Ala Ala
20 25 30
Ala Gly Ser Val Thr Arg His Tyr Lys Phe Asp Ile Lys Leu Lys Asn
35 40 45
Val Thr Arg Leu Cys Thr Thr Lys Ser Ile Val Thr Val Asn Gly Lys
50 55 60
Phe Pro Gly Pro Arg Ile Ile Ala Arg Glu Gly Asp Arg Val Leu Val
65 70 75 80
Asn Val Val Asn His Val Pro Asn Asn Met Thr Ile His Trp His Gly
85 90 95
Ile Arg Gln Leu Gln Asn Gly Trp Ala Asp Gly Pro Ser Tyr Ile Thr
100 105 110
Gln Cys Pro Ile Gln Thr Asn Gln Ser Tyr Val Tyr Asn Phe Thr Ile
115 120 125
Ile Gly Gln Arg Gly Thr Leu Phe Trp His Ala His Ile Ser Trp Leu
130 135 140
Arg Ala Thr Val Tyr Gly Pro Leu Ile Ile Leu Pro Lys Arg His Ala
145 150 155 160
Pro Tyr Pro Phe Pro Lys Pro His Lys Glu Val Pro Ile Ile Phe Gly
165 170 175
Glu Trp Trp Asn Val Asp Thr Glu Ala Val Ile Ser Gln Ala Leu Gln
180 185 190
Thr Gly Asn Gly Pro Asn Val Ser Asn Ala Tyr Thr Ile Asn Gly Leu
195 200 205
Pro Gly Pro Leu Tyr Asn Cys Ser Lys Lys Asp Thr Phe Ser Leu Lys
210 215 220
Val Lys Pro Gly Lys Thr Tyr Leu Leu Arg Leu Ile Asn Ala Ala Val
225 230 235 240
Asn Asp Glu Leu Phe Phe Ser Ile Ala Asn His Ser Leu Thr Val Val
245 250 255
Glu Ala Asp Ala Val Tyr Val Lys Pro Phe Thr Thr Asn Ile Leu Phe
260 265 270
Ile Thr Pro Gly Gln Thr Thr Asn Val Leu Leu Lys Ala Lys Pro Gln
275 280 285
Tyr Pro Asn Ala Thr Phe Leu Met Leu Ala Arg Pro Tyr Phe Thr Gly
290 295 300
Arg Gly Thr Phe Asp Asn Ser Thr Val Ala Gly Ile Leu Glu Tyr Lys
305 310 315 320
Glu His Ser Asn Ser Ser Ser Ser Thr Ser Leu Lys Ser Pro Leu Phe
325 330 335
Arg Pro Ile Leu Pro Gln Ile Asn Ala Thr Ser Phe Val Ala Asn Phe
340 345 350
Thr Ser Lys Phe Arg Ser Leu Ala Ser Ser Lys Phe Pro Ala Asn Val
355 360 365
Pro Arg Thr Val Glu Lys Arg Phe Phe Phe Thr Val Gly Leu Gly Thr
370 375 380
Asn Pro Cys Pro Lys Asn Gln Thr Cys Gln Gly Pro Thr Asn Thr Thr
385 390 395 400
Lys Phe Ala Ala Ser Ile Asn Asn Asn Ser Phe Ala Leu Pro Ser Val
405 410 415
Ala Leu Leu Gln Ser His Phe Phe Ala Gln Ser Asn Ser Val Tyr Thr
420 425 430
Thr Asp Phe Pro Thr Ile Pro Pro Lys Pro Phe Asn Tyr Thr Gly Thr
435 440 445
Pro Pro Asn Asn Thr Asn Val Ser Thr Gly Thr Lys Val Val Val Leu
450 455 460
Lys Phe Asn Thr Ser Val Glu Leu Val Leu Gln Gly Thr Ser Ile Leu
465 470 475 480
Gly Ala Glu Ser His Pro Leu His Leu His Gly Phe Asn Phe Phe Val
485 490 495
Val Gly Gln Gly Phe Gly Asn Phe Asp Pro Asn Lys Asp Pro Pro Lys
500 505 510
Phe Asn Leu Val Asp Pro Val Glu Arg Asn Thr Tyr Gly Val Pro Ala
515 520 525
Arg Gly Trp Ala Ala Ile Arg Phe Leu Ala Asp Asn Pro Gly Val Trp
530 535 540
Leu Met His Cys His Phe Asp Val His Leu Ser Trp Gly Leu Arg Met
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Pro Pro Ser Asp Leu Pro Lys Cys
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<210> 3
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atggcgtctt ctattccttt ttcac 25
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<400> 4
tcaacacttt gggagatcgg agc 23
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catgccatgg ccatggcgtc ttctattcct ttttcac 37
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gaagatctac actttgggag atcggacgg 29
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ggagaagaac ttttcactgg 20
<210> 8
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<212> DNA
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gtaatcccag cagctgttac 20
<210> 9
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<213> 人工序列
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tggcttttct tcttgctctt atctc 25
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cattgctgca gctggcagt 19
Claims (1)
1.一种梨漆酶基因PbLAC1在调控植物木质素合成及细胞壁发育中的应用,所述梨漆酶基因PbLAC1的核苷酸序列如SEQ ID NO.1所示,其特征在于,所述梨漆酶基因PbLAC1具有合成植物木质素、促进细胞壁增厚的作用;在植物体内超量表达该梨漆酶基因PbLAC1,提高植物茎干的刚性,增强抗倒伏能力;在梨果实中抑制该梨漆酶基因PbLAC1表达,抑制石细胞发育与果锈合成,提升梨果实的内在与外观品质;
其中,所述梨漆酶基因PbLAC1的克隆步骤如下:提取梨果实总RNA并反转录成cDNA,根据公布的梨基因组序列设计特异性扩增引物PbLAC1-F和PbLAC1-R,以cDNA为模板进行PCR扩增;其中,特异性扩增引物PbLAC1-F和PbLAC1-R的核苷酸序列分别如SEQ ID NO.3、SEQID NO.4所示;
所述梨漆酶基因PbLAC1超量表达载体及宿主细胞的构建方法如下:
(1)根据已经成功克隆的目的基因PbLAC1序列设计带有Bgl II和Nco I酶切位点的引物PbLAC1-ZH-F和PbLAC1-ZH-R,以已构建的pMD-18T-PbLAC1质粒为模板进行扩增,PCR产物经1%琼脂糖凝胶电泳检测后,对目的片段进行回收,并连接到载体pMD-18T上进行测序;其中,带有Nco I和Bgl II酶切位点的引物PbLAC1-ZH-F和PbLAC1-ZH-R的核苷酸序列分别如SEQ ID NO.5、SEQ ID NO.6所示;
(2)用限制性内切酶Bgl II和Nco I分别对pMD-18T-PbLAC1质粒和植物表达载体pCambia1304质粒进行双酶切;酶切产物用1%的琼脂糖凝胶电泳,回收目的片段PbLAC1和植物表达载体pCambia1304;用T4连接酶将2个酶切后的目的片段连接并转化到大肠杆菌DH5α感受态细胞,重组质粒进行酶切验证后并进行测序验证,最终获得植物超量表达载体pCambia1304-PbLAC1;
(3)将构建好的植物表达载体pCambia1304-PbLAC1通过电转化到农杆菌EHA105感受态细胞中,并通过卡那霉素和利福平筛选出阳性菌株,获得宿主细胞。
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