CN110923249A - 烟草CyP71及在调控植物表皮毛发育方面的应用 - Google Patents
烟草CyP71及在调控植物表皮毛发育方面的应用 Download PDFInfo
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Abstract
本发明公开了一种调控植物表皮毛发育的基因,其特征在于:该基因为烟草CyP71,其核苷酸序列如SEQ ID NO.1所示。本发明将CyP71构建到表达载体pSH737上后,在农杆菌LBA4404中扩繁。通过蘸花遗传转化法,将pSH737‑CyP71携带的CyP71转入植株中,获得转化植株。结果显示,转CyP71表皮毛数量较野生型拟南芥多增加了100%,表明表达CyP71的蛋白具有正调控植物表皮毛发育,实现了增加植物表皮毛的数量。
Description
技术领域
本发明涉及基因工程领域,特别是涉及一种烟草—CyP71,同时还涉及该烟草CyP71在调控植物表皮毛发育方面的应用。
背景技术
表皮毛是由植物表皮细胞发育的突出植物表皮的多细胞结构,也被称为毛被,作为植物组织表面的表皮毛,它通过增加植物表皮层的厚度来减少水分散失和调节植物表面的温度。植物腺毛对植物抗逆境胁迫、植物病虫害防御等起重要作用,非分泌型腺毛构成植物的第一道防线,分泌型腺毛通过分泌挥发性的萜类等来抵御病虫害,并且分泌的二萜类化合物能增加叶面的粘性,达到捕杀昆虫的效果。
一些植物的表皮毛具有特殊的经济价值,例如棉花的棉纤维、蕨类植物蜈蚣草的表皮毛、烟草的烟叶腺毛和拟南芥的表皮毛等。棉纤维由棉花种子的表皮细胞分化发育形成的单细胞纤维组成,棉纤维是纺织工业重要的天然纤维。蕨类植物蜈蚣草的表皮毛能大量吸收重金属砷。烟草腺毛主要产生代谢分泌物,这些代谢分泌物能够影响植物本身的生长。类萜代谢途径是分泌型腺毛最活跃的代谢途径之一,分泌的萜类化合物不仅能提升烟草的商业价值,同时也能形成化学防御体系。拟南芥的表皮毛是典型的特化无腺体的单细胞表皮毛,分布于整个叶片、茎和花瓣上。拟南芥表皮毛作为一种特化的、典型的单细胞结构,可作为一种很好的模式系统来研究细胞命运决定,细胞周期调控、细胞极性以及细胞增大等细胞分化方面的问题。
拟南芥已成为全球应用最广泛的模式植物,被誉为“植物中的果蝇”,其植株较小,生长周期短,结实多且易于转化。植物进化发育出表皮毛不仅可以发挥其特定的生理功能即适应不利的环境,还具有特殊的经济价值。
随着分子生物学技术的发展,通过基因工程手段,利用调控表皮毛发育的关键基因,将为植物表皮毛发育分子调控提供技术支撑,从而提高植物表皮毛的商业价值以及对外界环境的抗逆性和抗虫性。
发明内容
本发明的目的在于克服上述缺点而提供的一种能调控植物表皮的发育,且快速、高效地实现植物表皮毛数量增加的利用烟草CyP71。
本发明的再一个目的是提供该烟草CyP71在调控植物表皮毛发育方面的应用。
本发明的技术方案:一种调控植物表皮毛发育的基因,该基因为烟草CyP71,其核苷酸序列如SEQ ID NO.1所示。
根据权利要求1所述的一种调控植物表皮毛发育的基因,所述的CyP71,其克隆正向引物为5'-ATGCAACTGAGATTTGAAG-3',反向引物为5'- TCACTTTTGAGGAGGTTG-3'。
一种调控植物表皮毛发育的基因的植物表达载体的构建及遗传转化方法,包含以下步骤:(1)以测序正确的烟草CyP71基因T克隆重组质粒为模板,酶切引物扩增,扩增产物通过琼脂糖凝胶电泳检测,回收纯化;(2)对酶切引物扩增产物和pSH737进行双酶切,37℃恒温水浴酶切反应1.5h,酶切完成对产物进行电泳检测并切胶回收目的片段;(3)pSH737表达载体和酶切引物扩增产物的双酶切产物在T4连接酶的作用下,于恒温水浴16℃连接10h,连接为重组表达载体;(4)将连接产物转化到农杆菌LBA4404,并在其中扩增,选择Kan LB进行抗性筛选,然后进行菌落PCR验证其为阳性重组菌落,提取质粒并进行质粒PCR验证,并测序,成功得到pSH737-CyP71。
所述的酶切引物为:上游引物序列为5'-GCTCTAGAATGCAACTGAGATTTGAAG -3',下游引物序列为5'-TCCCCCGGGTTATCACTTTTGAGGAGGTTG -3'。
所述的双酶切采用Xba I和Sma I进行。
所述的烟草CyP71基因T克隆重组质粒的合成方法:分别利用CyP71克隆引物通过RT-PCR的方法从烟草中扩增基因,将扩增获得的PCR产物直接按照TA克隆方法克隆到克隆中间载体:pGEM-T上,在T4 DNA连接酶的作用下,将PCR产物与T载体充分混匀,于16℃连接过夜,将连接产物转化到大肠杆菌DH5α,并在其中扩增,随后进行菌落PCR和质粒PCR筛选阳性克隆,并测序即可。
所述的RT-PCR反应程序为: 50℃ 30min,94℃ 2min,94℃ 30S,55-65℃ 30S,72℃ 1min/kb,30个循环。
本发明与现有技术相比,具有明显的有益效果,从以上技术方案可知:本发明将CyP71构建到表达载体pSH737上后,在农杆菌LBA4404中扩繁。通过蘸花遗传转化法,将pSH737-CyP71携带的CyP71转入植株中,获得转化植株。结果显示,转CyP71表皮毛数量较野生型拟南芥多增加了100%,表明表达CyP71的蛋白具有正调控植物表皮毛发育,实现了增加植物表皮毛的数量。因此,CyP71及其编码蛋白可以调节植物表皮毛的发育,且快速、高效地实现植物表皮毛数量的增加,从而提高植物表皮毛的利用价值以及对外界环境的抗逆性和抗虫性。
附图说明
图1为克隆中间载体pGEM-T的结构示意图;
图2为植物表达载体pSH737的结构示意图;
图3为转CyP71拟南芥抗性植株的基因组PCR鉴定;
图4为GUS组织化学染色检测抗性植株;
图5为转CyP71拟南芥和野生型拟南芥表皮毛密度的比较。
具体实施方式
实施例1
(1)烟草CyP71的筛选
从烟草(Nicotiana tabacum cv.Xanthin)品种中通过提取野生型烟草总RNA,采用Agilent公司的烟草表达谱4*44K芯片,检测烟草全基因表达谱,根据芯片结果对基因的功能注释,结合KEGG、GO分析,筛选出1个与腺毛发育的关键基因:CyP71;
(2)烟草CyP71的克隆
分别利用 KC4800444.1的CyP71克隆正向引物5'-ATGCAACTGAGATTTGAAG-3'和CyP71克隆反向引物5'-TCACTTTTGAGGAGGTTG-3'通过RT-PCR的方法从烟草(Nicotiana tabacum cv. Xanthin)中扩增基因。
RT-PCR反应程序为: 50℃ 30min,94℃ 2min,94℃ 30S,55-65℃ 30S,72℃1min/kb,30个循环。
CyP71克隆载体的获得:将扩增获得的PCR产物直接按照TA克隆方法克隆到如图3所示的克隆中间载体:pGEM-T上。在T4 DNA连接酶的作用下,将PCR产物与T载体充分混匀,于16℃连接过夜。将连接产物转化到大肠杆菌DH5α,并在其中扩增,随后进行菌落PCR和质粒PCR筛选阳性克隆,并测序。
(3)烟草CyP71的植物表达载体的构建及遗传转化
目的DNA片段的酶切引物扩增。根据目的基因的酶切位点分析结果、pSH737所含的酶切位点以及内切酶之间的双酶切活性等方面综合考虑,分别利用XbaI和SmaI进行对双酶切,合成两端添加酶切位点的引物。根据pSH737所含的酶切位点设计CyP71上游引物序列为5'-GCTCTAGAATGCAACTGAGATTTGAAG -3',下游引物序列为5'-TCCCCCGGGTTATCACTTTTGAGGAGGTTG -3'。以测序正确的T克隆重组质粒为模板,酶切引物扩增,扩增产物通过1%琼脂糖凝胶电泳检测,同时回收纯化,经核酸分析仪分析检测浓度。对酶切引物扩增产物和pSH737进行双酶切,37℃恒温水浴酶切反应1.5h,酶切完成对产物进行电泳检测并切胶回收目的片段。表达载体和外源DNA的双酶切产物会形成线性互补的粘性末端,在T4连接酶的作用下,粘性末端互补配对,连接成为一个新的核酸分子,即为重组表达载体,于恒温水浴16℃连接10h。将连接产物转化到农杆菌LBA4404,并在其中扩增,由于pSH737带有Kan抗性基因,选择Kan LB进行抗性筛选,然后进行菌落PCR验证其为阳性重组菌落,提取质粒并进行质粒PCR验证,并测序。
将CyP71构建在pSH737-CyP71上,用于在植物中过表达,研究其功能。拟南芥的遗传转化方法采用蘸花法进行。植物中的筛选标记为Kan和Rif,从而获得烟草CyP71的转化植株。
试验例1:烟草CyP71在调控植物表皮毛发育方面的应用
(1)抗性植株的筛选和鉴定
转化野生型拟南芥获得的种子经Kan 100mg/L的抗性筛选1/2MS培养基筛选,抗性植株在筛选平板上能够正常生长,可以明显区别于白化的非抗性幼苗。待长到开花期后利用植物基因组提取试剂盒提取随机挑选的CyP71抗性植株基生叶基因组DNA,并进行PCR验证及 GUS组织化学染色鉴定。植物的表达载体pSH737的GUS报告基因位于35S启动子后,如果外源基因在拟南芥中表达,则会激活报告基因,所以使用GUS染色验证拟南芥抗性植株是否为转基因植株。剪取抗性拟南芥植株基生叶进行GUS染色:将叶片和GUS染色液置于PCR管中,37℃恒温培养箱避光培养12-16h,依次用75%乙醇溶液、95%乙醇和无水乙醇相继脱色处理并在体视镜下观察染色情况。GUS染色结果表明:叶片的表皮毛细胞被着色,表皮毛染上蓝色,表明叶片GUS报告基因能够启动表达,CyP71成功转入到植物中并表达;
(2)转CyP71拟南芥和野生型拟南芥表皮毛数量差异比较
在体式显微镜低倍镜下对比表皮毛性状差异。选取开花期(10周)野生型拟南芥和转CyP71拟南芥基生叶各3片,在体式显微镜1*2放大倍数下,随机抽取8个视野(实际面积24mm2)进行表皮毛数量统计并绘制图表。结果显示:转CyP71拟南芥的表皮毛较野生型拟南芥表皮毛数量提高至100%,表明CyP71能够提高植物表皮毛数量。
以上所述,仅是本发明的实施例而已,并非对本发明作任何形式上的限制,任何未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
序列表
<110>贵州省烟草科学研究院
<120> 烟草CyP71及在调控植物表皮毛发育方面的应用
<130> 1
<160> 4
<210> 1
<211> 1545
<212> DNA
<213> CYP71
<400> 1
ATGCAACTGAGATTTGAAGAATACCAACTAACCAAAATGCAGTTCTTCAGCTTGGTTTCC 60
ATTTTCCTATTTCTATCTTTCCTCTTTTTGTTAAGGATATGGAAGAACTCCAATAGCCAA 120
AGCAAAAAGTTGCCACCAGGTCCATGGAAACTACCAATACTAGGAAGTATGCTTCATATG 180
GTTGGTGGACTACCACACCATGTCCTTAGAGATTTAGCCAAAAAATATGGACCACTTATG 240
CACCTTCAATTAGGTGAAGTTTCTGCGGTTGTGGTTACTTCTCCTGATACGGCAAAAGAA 300
GTATTAAAAACTCATGACATCGCTTTTGCGTCTAGGCCTAGCCTTTTGGCCCCGGAGATT 360
GTCTGTTACAATAGGTCTGATCTAGCCTTTTGCCCCTATGGCGACTATTGGAGACAAATG 420
CGTAAAATATGTGTCTTGGAAGTGCTCAGTGCCAAGAATGTTCGGACATTTAGCTCTATT 480
AGGCGGAATGAAGTTCTTCGTCTCATTAATTTTATCCGGTCATCTTCTGGTGAACCTATT 540
AATGTTACGGAAAGGATCTTTTTGTTCACAAGCTCCATGACATGTAGATCAGCGTTTGGG 600
CAAGTGTTCAAAGAGCAAGACAAATTTATACAACTAATTAAAGAAGTGATACTCTTAGCA 660
GGAGGGTTTGATGTGGCTGACATATTCCCTTCACTGAAGTTTCTTCATGTGCTCAGTGGA 720
ATGAAGGGTAAGATTATGAATGCACACCATAAGGTAGATGCCATTGTTGAGAATGTCATC 780
AATGAGCACAAGAAAAATCTTGCAATTGGGAAAACTAATGGAGCGTTAGGAGGTGAAGAT 840
TTAATTGATGTTCTTCTAAGACTTATGAATGATGGAGGCCTTCAATTTCCTATCACCAAC 900
GACAACATCAAAGCTATAATTTTTGACATGTTTGCTGCCGGGACAGAGACTTCATCGTCA 960
ACAATTGTGTGGGCTATGGTAGAAATGGTGAAAAATCCAGCCGTATTCGCGAAAGCTCAA 1020
GCAGAAGTAAGAGAAGCATTTAGAGGAAAAGAAACTTTCGATGAAAATGATGTGGAGGAG 1080
CTAAACTACCTAAAGTTAGTAATAAAAGAAACTCTAAGACTTCATCCACCGGTTCCACTT 1140
TTGCTCCCAAGAGAATGTAGGGAAGAGACAAATATAAACGGCTACACTATTCCTGTAAAG 1200
ACCAAAGTCATGGTTAATGTTTGGGCTTTGGGAAGAGATCCAAAATATTGGAATGACGCA 1260
GAAACTTTTATGCCAGAGAGATTTGAGCAGTGCTCTAAGGATTTTGTTGGTAATAATTTT 1320
GAATATCTTCCATTTGGTGGCGGAAGGAGGATTTGTCCTGGGATTTCGTTTGGCTTAGCT 1380
AATGCTTATTTGCCATTGGCTCAATTACTATATCACTTCGATTGGAAACTCCCTGCTGGA 1440
ATCGAACCAAGCGACTTGGACTTGACTGAGTTGGTTGGAGTAACTGCCGCTAGAAAAAGT 1500
GACCTTTACTTGGTTGCGACTCCTTATCAACCTCCTCAAAAGTGA 1545
<210> 2
<211> 514
<212> 氨基酸
<213> CYP71
<400> 2
MQLRFEEYQLTKMQFFSLVSIFLFLSFLFLLRIWKNSNSQSKKLPPGPWKLPILGSMLHM 60
VGGLPHHVLRDLAKKYGPLMHLQLGEVSAVVVTSPDTAKEVLKTHDIAFASRPSLLAPEI 120
VCYNRSDLAFCPYGDYWRQMRKICVLEVLSAKNVRTFSSIRRNEVLRLINFIRSSSGEPI 180
NVTERIFLFTSSMTCRSAFGQVFKEQDKFIQLIKEVILLAGGFDVADIFPSLKFLHVLSG 240
MKGKIMNAHHKVDAIVENVINEHKKNLAIGKTNGALGGEDLIDVLLRLMNDGGLQFPITN 300
DNIKAIIFDMFAAGTETSSSTIVWAMVEMVKNPAVFAKAQAEVREAFRGKETFDENDVEE 360
LNYLKLVIKETLRLHPPVPLLLPRECREETNINGYTIPVKTKVMVNVWALGRDPKYWNDA 420
ETFMPERFEQCSKDFVGNNFEYLPFGGGRRICPGISFGLANAYLPLAQLLYHFDWKLPAG 480
IEPSDLDLTELVGVTAARKSDLYLVATPYQPPQK 514
<210> 3
<211> 19
<212> DNA
<213> 人工合成
<400> 3
ATGCAACTGAGATTTGAAG
<210> 4
<211> 18
<212> DNA
<213> 人工合成
<400> 4
TCACTTTTGAGGAGGTTG
<210> 5
<211> 27
<212> DNA
<213> 人工合成
<400> 5
GCTCTAGAATGCAACTGAGATTTGAAG
<210> 6
<211> 30
<212> DNA
<213> 人工合成
<400> 6
TCCCCCGGGTTATCACTTTTGAGGAGGTTG
Claims (7)
1.一种调控植物表皮毛发育的基因,其特征在于:该基因为烟草CyP71,其核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的一种调控植物表皮毛发育的基因,其特征在于:所述的CyP71,其克隆正向引物为5'-ATGCAACTGAGATTTGAAG-3',反向引物为5'-TCACTTTTGAGGAGGTTG-3'。
3.如权利要求1或2所述的一种调控植物表皮毛发育的基因的植物表达载体的构建及遗传转化方法,其特征在于:包含以下步骤:(1)以测序正确的烟草CyP71基因T克隆重组质粒为模板,酶切引物扩增,扩增产物通过琼脂糖凝胶电泳检测,回收纯化;(2)对酶切引物扩增产物和pSH737进行双酶切,37℃恒温水浴酶切反应1.5h,酶切完成对产物进行电泳检测并切胶回收目的片段;(3)pSH737表达载体和酶切引物扩增产物的双酶切产物在T4连接酶的作用下,于恒温水浴16℃连接10h,连接为重组表达载体;(4)将连接产物转化到农杆菌LBA4404,并在其中扩增,选择Kan LB进行抗性筛选,然后进行菌落PCR验证其为阳性重组菌落,提取质粒并进行质粒PCR验证,并测序,成功得到pSH737-CyP71。
4.根据权利要求3所述的一种调控植物表皮毛发育的基因的植物表达载体的构建及遗传转化方法,其特征在于:所述的酶切引物为:上游引物序列为5'-GCTCTAGAATGCAACTGAGATTTGAAG -3',下游引物序列为5'-TCCCCCGGGTTATCACTTTTGAGGAGGTTG -3'。
5.根据权利要求3所述的一种调控植物表皮毛发育的基因的植物表达载体的构建及遗传转化方法,其特征在于:所述的双酶切采用XbaI和SmaI进行。
6.根据权利要求3所述的一种调控植物表皮毛发育的基因的植物表达载体的构建及遗传转化方法,其特征在于:所述的烟草CyP71基因T克隆重组质粒的合成方法:分别利用CyP71克隆引物通过RT-PCR的方法从烟草中扩增基因,将扩增获得的PCR产物直接按照TA克隆方法克隆到克隆中间载体:pGEM-T上,在T4 DNA连接酶的作用下,将PCR产物与T载体充分混匀,于16℃连接过夜,将连接产物转化到大肠杆菌DH5α,并在其中扩增,随后进行菌落PCR和质粒PCR筛选阳性克隆,并测序即可。
7.根据权利要求6所述的一种调控植物表皮毛发育的基因的植物表达载体的构建及遗传转化方法,其特征在于:所述的RT-PCR反应程序为: 50℃ 30min,94℃ 2min,94℃ 30S,55-65℃ 30S,72℃ 1min/kb,30个循环。
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