CN111621520A - 一种马尾松PmCOMT基因的应用 - Google Patents
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Abstract
本发明公开了一种马尾松PmCOMT基因的应用,属于植物基因工程技术领域。本发明为首次利用马尾松PmCOMT基因定向促进植物中S木质素单体合成,通过构建含有马尾松PmCOMT基因的载体并将其转化到农杆菌感受态细胞中,再侵染烟草,得到转PmCOMT基因型烟草,检测结果显示转PmCOMT基因型烟草木质部中的S木质素较野生型多,转基因烟草植株中S木质素含量比野生型烟草提高10%以上,显著差异,表明本发明有效地提高了植物中S木质素单体合成,调控了植物中木质素S/G比值。本发明有助于对马尾松PmCOMT基因功能的深入研究,并推动马尾松转基因定向调控木质素合成的基因工程育种的应用。
Description
技术领域
本发明属于植物基因工程技术领域,更具体地说,涉及一种马尾松PmCOMT基因的应用。
背景技术
马尾松(Pinus massoniana Lamb.)是松科松属多年生常绿树种,是我国特有的乡土树种,其适应性强,耐干旱瘠薄,广泛分布在秦岭以南18个省,主产地区为赣、浙、闽、桂、粤、湘、川、渝、鄂、黔等地。马尾松林面积与木材蓄积量均居我国林木前列,其木材纤维细长,非常适合造纸,因而马尾松是我国主要的纸浆材树种。由于天然木材中纤维素和木质素紧密结合,去除木质素的过程是纸浆生产过程中一个重要环节,因此需求低木质素含量的木材。但从植物生物学角度出发,木质素在植物正常生长发育和病虫害抗性方面具有非常重要的功能,植物体内木质素含量大量减少对植物生长具有很大的影响。因此,在不大幅降低木质素总量前提下,调控不同木质素单体比例,提高木质素S/G比值,是纸浆材定向育种的主要策略。
咖啡酸-O-甲基转移酶基因(Caffeic-acid-O-methyltransferase,COMT)是植物木质素合成途径中的关键基因之一,其表达产物咖啡酸-O-甲基转移酶是木质素单体合成的关键酶之一。针对植物COMT基因及其功能的研究发现,对玉米COMT基因进行抑制后发现S木质素含量显著降低。在高度多倍体甘蔗中利用TALEN介导的多COMT等位基因突变,提高了田间甘蔗木质素的还原率,显著降低了S/G木质素含量比值并提高了糖化效率。利用RNAi技术降低大麦中COMT活性,降低了木质素总量的同时还显著降低了S/G木质素比值,还减少了木质素与细胞壁的连接程度,在COMT-RNAi介导的柳枝稷中也有类似的现象。这些结果表明抑制COMT表达会降低S木质素的合成,并降低S/G比值,说明S木质素的合成与S/G的比值都受到COMT基因的调控。本发明公开的马尾松COMT序列及其在基因工程中的应用,有助于促进马尾松木质素合成调控的基因工程定向育种。
发明内容
针对现有技术存在的上述问题,本发明所要解决的技术问题在于提供一种马尾松PmCOMT基因在提高植物中S木质素含量中的应用,能有效地提高植物中S木质素单体的合成。
为了解决上述技术问题,本发明所采用的技术方案如下:
一种马尾松PmCOMT基因在提高植物中S木质素含量中的应用,是将马尾松PmCOMT基因转化到植物或植物细胞中,获得S木质素含量提高的转基因植物。
进一步地,所述应用的具体步骤为:
1)构建含有马尾松PmCOMT基因的载体;
2)使用步骤1)得到的载体转化宿主细胞;
3)使用步骤2)得到的宿主细胞侵染植物或植物细胞;
4)筛选得到S木质素含量提高的转基因植物。
进一步地,所述的载体为植物表达载体。
进一步地,所述的植物表达载体具体为pCAMBIA-1302-COMT。
进一步地,所述的宿主细胞为农杆菌。
进一步地,所述的植物为烟草。
相比于现有技术,本发明的有益效果为:
(1)本发明为首次利用马尾松PmCOMT基因定向促进植物中S木质素单体合成,通过构建含有马尾松PmCOMT基因的载体并将其转化到农杆菌感受态细胞中,再侵染烟草,检测结果显示转PmCOMT基因型烟草木质部中的S木质素较野生型多,转基因烟草植株中S木质素含量比野生型烟草提高10%以上,显著差异,表明本发明有效地提高了植物中S木质素单体合成,调控了植物中木质素S/G比值;
(2)本发明有助于对马尾松PmCOMT基因功能的深入研究,并推动马尾松转基因定向调控木质素合成的基因工程育种的应用。
附图说明
图1为PmCOMT重组蛋白上清液的SDS-PAGE凝胶电泳结果图;注:Marker:PAGE-MASTER蛋白质Marker;1:1mM IPTG诱导pET28a空载蛋白;2:未诱导PmCOMT重组蛋白;3:0.2mM IPTG诱导PmCOMT重组蛋白;4:0.4mMIPTG诱导PmCOMT重组蛋白;5:0.6mM IPTG诱导PmCOMT重组蛋白;6:0.8mM IPTG诱导PmCOMT重组蛋白;7:1mM IPTG诱导PmCOMT重组蛋白;8:2mM IPTG诱导PmCOMT重组蛋白
图2为LC-MS测定PmCOMT的底物反应结果图;
图3为T1代烟草电泳检测结果图;
图4为烟草茎中木质素的分布检测结果图;注:A:空白对照,B:1302-PmCOMT转基因烟草。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。下述实施例中的%,如无特殊说明,均为质量百分含量。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
实施例1:马尾松PmCOMT基因片段的获得
取7年生马尾松木质部组织,利用植物总RNA提取试剂盒(TIANGEN)提取总RNA,利用逆转录试剂盒合成cDNA(TIANGEN),按照试剂盒提供的说明书操作。利用引物COMT-ORF-F和COMT-ORF-R(序列COMT-ORF-F:5′-ATGGATTGTAAGGGTAACAACAGC-3′;COMT-ORF-R:5′-TTTCTGAATTTCTAGAAGACTGTAGAAAG-3′),以及2×高保真酶Mix和cDNA模板PCR扩增出PmCOMT基因片段。PCR扩增产物经过电泳检测并回收后,连入克隆载体pEASY(TransGen)并转化大肠杆菌T1菌株感受态细胞,经过抗生素筛选和菌落PCR鉴定后将阳性菌进行测序,将连接好的克隆载体命名为pEASY-COMT。获得PmCOMT基因编码蛋白质的ORF序列(GenBanK登陆号:MH059810)。利用ORFfinder对PmCOMT的编码区进行翻译成氨基酸序列。
实施例2:马尾松COMT基因正义表达载体构建
利用Nco I限制性内切酶在CaMV 35S强启动子下游对植物表达载体pCAMBIA-1302进行酶切,并回收大片段线性化质粒。利用含有同源重组序列的引物COMT-CZ-F和COMT-CZ-R(序列COMT-CZ-F:5′-GGACTCTTGACATGGATTGTAAGGGTAACAACAGC-3′;COMT-CZ-R:5′-GTCAGATCTACTTTCTGAATTTCTAGAAGACTGTAGAAAG-3′),以及2×高保真酶Mix和上述含有pEASY-COMT载体的大肠杆菌作为模板PCR扩增出含有重组臂序列的PmCOMT基因片段,PCR扩增产物经过电泳检测并回收。
利用重组酶(诺唯赞)将线性化的pCAMBIA-1302载体和含有重组臂序列的PmCOMT基因片段连接,按照试剂供应商提供的说明书操作。将重组连接的载体转化大肠杆菌T1菌株感受态细胞,经过抗生素筛选和菌落PCR鉴定后将阳性菌进行测序,并将构建好载体命名为pCAMBIA-1302-COMT。
实施例3:马尾松COMT重组蛋白的获得
利用COMT-DB-F和COMT-DB-R(序列COMT-DB-F:5′-GGGTCGCGGATCCGAATTCATGGATTGTAAGGGTAACAAC-3′;COMT-DB-R:5′-CTCGAGTGCGGCCGCAAGCTTTTTCTGAATTTCTAGAAGACTG-3′),以及2×高保真酶Mix和上述含有pEASY-COMT载体的大肠杆菌作为模板PCR扩增出不含有终止密码子的PmCOMT基因片段,PCR扩增产物经过电泳检测并回收,利用重组蛋白表达载体构建试剂盒(TransGen)和该回收的基因片段构建用于原核表达马尾松COMT重组蛋白的载体,并转化大肠杆菌T1菌株感受态细胞,经过抗生素筛选和菌落PCR鉴定后将阳性菌进行测序,并命名为pE2-COMT。
利用质体小提试剂盒(TIANGEN)从上述含有pEASY-COMT载体的大肠杆菌提取pEASY-COMT载体,并转化大肠杆菌Transetta(DE3)菌株表达感受态细胞,经过抗生素筛选和菌落PCR鉴定后,将单菌落接种至50mL含有抗生素和终浓度为0.2mM、0.4mM或0.6mM的IPTG的LB液体培养基中,在37℃,200rpm的条件下在摇床上摇菌8h。将发酵好的菌液离心10min收集菌体,并利用超声破碎并离心后收集上清,进行SDS-PAGE电泳检测,结果如图1所示。
利用Ni-NTA蛋白纯化试剂盒并根据说明书操作,纯化出上述上清液中的马尾松COMT重组蛋白。将5mL蛋白液与终浓度为500μM的咖啡酸底物在37℃条件下反应6h,并通过乙酸乙酯萃取反应物后进行液相色谱-质谱检测,可检测到酶促反应产物阿魏酸的特征峰,结果如图2所示。
实施例4:利用马尾松COMT基因调控烟草木质素单体合成
利用质粒小提试剂盒(TIANGEN)提取上述pCAMBIA-1302-COMT载体并转化到农杆菌EHA105菌株感受态细胞,经过抗生素筛选和菌落PCR鉴定后,按照常规方法利用农杆菌菌液浸染切割后的红花烟草叶片,获得待检测转PmCOMT基因的烟草苗。
1)转基因烟草PCR鉴定
采集移栽4周后待检测烟草叶片提取基因组DNA,用引物COMT-CZ-F和COMT-CZ-R对提取的基因组DNA进行PCR鉴定,可以扩增出约1100bp条带的烟草苗认为是阳性转PmCOMT基因烟草,如图3所示。
2)木质素分布检测
通过Maüile反应检测转PmCOMT烟草及野生烟草的茎段,如图4所示。其横切面可观察到S木质素被染色成紫红色(图4中深色箭头所指),G木质素被染成黄色(图4中浅色箭头所指),转PmCOMT型与野生型烟草茎段中S木质素多沉降于木质部的次生细胞壁中,G木质素多沉降于韧皮部的次生细胞壁中,初步可见转PmCOMT基因型烟草木质部中的S木质素较野生型多,如图4所示。
3)木质素单体含量检测
截取10月龄转PmCOMT基因型和野生型烟草茎端下第5节至第15节茎段,制成小段至于65℃烘干24h,粉碎。利用硫代酸解法反应后加入内标二十四烷溶于CH2C12并取有机进行相气相色谱/质谱联用分析,结果显示,转PmCOMT基因型烟草中S木质素含量为9.30±0.54,野生型烟草中S木质素含量为8.36±0.44,转基因型较野生型高11.24%,差异显著。
Claims (6)
1.一种马尾松PmCOMT基因在提高植物中S木质素含量中的应用,其特征在于,将马尾松PmCOMT基因转化到植物或植物细胞中,获得S木质素含量提高的转基因植物。
2.根据权利要求1所述的应用,其特征在于,所述应用的具体步骤为:
1)构建含有马尾松PmCOMT基因的载体;
2)使用步骤1)得到的载体转化宿主细胞;
3)使用步骤2)得到的宿主细胞侵染植物或植物细胞;
4)筛选得到S木质素含量提高的转基因植物。
3.根据权利要求2所述的应用,其特征在于,所述的载体为植物表达载体。
4.根据权利要求3所述的应用,其特征在于,所述的植物表达载体具体为pCAMBIA-1302-COMT。
5.根据权利要求2所述的应用,其特征在于,所述的宿主细胞为农杆菌。
6.根据权利要求1~5任一所述的应用,其特征在于,所述的植物为烟草。
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