CN112877355A - 一种利用烟草表达三七皂苷的方法 - Google Patents
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Abstract
本发明公开一种利用烟草表达三七皂苷的方法,属于生物技术领域。本发明所述方法为:参照三七的基因组序列,在两端分别设计正反引物,克隆三七中的SS基因;将SS基因构建到植物表达载体pCAMBIA2300‑35S,并命名为pCAMBIA2300‑35S‑SS2‑1,将其转化到农杆菌EHA105中,利用农杆菌介导的方法把含SS基因的质粒转化烟草,当转基因烟草生长一段时间后采集叶片,用甲醇提取烟叶中的皂苷,对烟草叶片中单体皂苷Rb1,Rd,R1,Re,Rg1含量进行检测;结果显示,野生型烟草总未检测出皂苷,而在转基因烟草植株中检测到Rg1,Re,Rb1共3种单体皂苷。
Description
技术领域
本发明涉及一种利用烟草表达三七皂苷的方法,属于生物技术领域。
背景技术
三七Panaxnotoginseng(Burk.)F.H.Chen是五加科人参属的草本植物,三七总皂苷是三七主要的药用活性成分,对治疗心脑血管疾病、胃肠道疾病、抑制癌细胞等方面效果显著。三七总皂苷为达玛烷型四环三萜皂苷,鲨烯是所有三萜皂苷及甾醇等萜烯类物质的共同前体,因此对于催化鲨烯合成的鲨烯合酶(squalene synthase,SS)基因的研究,有助于促进三七总皂苷的合成。鲨烯合酶(squalene synthase,SS)是生成三萜化合物和植物甾醇的前体,它的作用在于能使法呢基焦磷酸(farnesyldiphosphate,FPP)缩合生成鲨烯。增加植物中SS基因的含量和活性,能够有效促进植株中三萜皂苷的合成。有研究表明,过表达SS基因会使该途径下游其它酶的含量和活性增加,从而提高人参皂苷的产量。
随着组培技术和转基因技术体系逐渐成熟,烟草作为模式植物在生命科学研究领域发挥重要作用。并且烟草具有易培养、易种植、生长周期短、可收获大量种子等特点,十分有利于转化SS基因。转基因烟草还因其安全、环保,生长迅速的特性成为生产生物制品的理想载体,我们将皂苷合成关键酶基因转化烟草,希望利用烟草这个理想的生产生物制品的载体,可以大量提取三七皂苷。从而打破三七生产上的生长周期慢、易造成病害及连作障碍等制约三七皂苷生产的难题。
发明内容
本发明的目的在于提供一种利用烟草表达三七皂苷的方法,具体包括以下步骤:
(1)参照三七的基因组序列,在两端分别设计正反引物,克隆三七中的SS基因序列如SEQ ID NO:1;
所述正反引物分别是SS FP:5’-ACGCGTCGACATGGGAAGTTTGGGGGCAATTC-3’,SSRP:5’-TGGTTCTGCAGTCACTGTTTGTTCGGTAGTAG-3’;
(2)将三七SS基因克隆到植物表达载体中,使三七SS基因在35S启动子及其它启动子的作用下进行基因转录与表达;
(3)利用三七完整或部分的SS基因(CDS序列及基因组序列)转化烟草,使三七SS基因在烟草包括烟草中表达进行皂苷合成。
本发明步骤(2)中所述所述植物表达载体为pCAMBIA2300,但并不限于pCAMBIA2300,其他满足要求的植物载体也可用于本发明。
将SS基因构建到植物表达载体pCAMBIA2300-35S中,并命名为pCAMBIA2300-35S-SS2-1,将其转化到农杆菌EHA105中,利用农杆菌介导的方法把含SS基因的质粒转化烟草。
优选的,本发明所述表达载体pCAMBIA2300-35S的构建方法为:先将35S-GFP-terminator插入pCAMBIA2300载体上,插入多克隆位点5’EcoRI/HindIII3’之间;构建pCAMBIA2300-35S-GFP载体,再用5’SalI--PstI 3’切开GFP部分,插入SS基因替换原载体的GFP基因。优选的,本发明所述表达载体pCAMBIA2300-35S的构建方法为:先将35S-GFP-terminator插入pCAMBIA2300载体上,插入多克隆位点5’EcoRI/HindIII 3’之间;构建pCAMBIA2300-35S-GFP载体,再用5’SalI--PstI 3’切开GFP部分,插入SS基因替换原载体的GFP基因。
本发明所述三七SS基因在烟草包括烟草品种巴斯玛14号或其它烟草品种中表达。
优选的,本发明步骤(2)利用农杆菌介导的方法把含SS基因的质粒转化烟草的具体过程为:在超净工作台将烟叶剪成0.5cm2大小,放入农杆菌浸染液中浸泡3min,取出烟叶,用滤纸吸取多余浸染液后,将叶片转入含有0.5mg/L浓度的6-BA培养基上,黑暗培养3天;经过黑暗培养的烟叶转入含有为500mg/L羧苄青霉素的MS培养基上,25℃±1℃,光照培养30至45天;形成愈伤组织后,嫩芽陆续从中长出,将组织整体转入含有羧苄青霉素和浓度为50mg/L卡那霉素的MS选择压培养基上,25℃±1℃,光照培养7至10天;经过筛选后,继续生长的烟芽从愈伤组织上分离下来,转至生根培养基中,25℃±1℃,光照培养;生根的烟草苗长至4-5cm高,有3到4片小叶时就可炼苗移栽。
本发明的有益效果:
(1)本发明所述方法成功从三七基因组中克隆出SS基因,并构建到表达载体pCAMBIA2300中,将SS基因转入烟草中,可以大量提取三七皂苷。鲨烯合成酶(squalenesynthase,SS)既是三萜和甾醇分支代谢途径中的第1个酶,也是第1个关键酶,对植物甾醇和三萜生物合成均起着重要调控作用。以植物作为生物反应器可以大大降低生产成本,简化储存方式。
(2)本方法成功将三七鲨烯合酶基因转化烟草组培苗,获得转基因植株,并且检测到转基因烟草叶片中含有3种单体皂苷;烟草具有生长周期短,易栽种等特点,可以从转基因烟草中提取皂苷,用于医药制品,从而打破三七生产上的生长周期慢、易造成病害及连作障碍等制约三七皂苷生产的难题。
附图说明
图1为表达载体图。
图2表达载体pCAMBIA2300-35S-SS2-1PCR与酶切结果,注:M-Marker;1、2:PCR结果;3、4:酶切结果。
图3烟草农杆菌转化法;A-组培苗;B-共培养期;C-除菌培养期;D-愈伤形成期;E-筛选培养期;F-生根阶段。
图4烟草移栽;
图5转基因烟草PCR检测;M-Marker;1-负对照2-野生型烟草;3-转基因烟草;4-正对照;
图6野生型烟草皂苷含量检测;
图7转SS基因烟草皂苷含量检测。
具体实施方式
下面结合具体实施例本发明作进一步的详细说明,但本发明的保护范围并不限于所述内容。
实施例1
本发明实施例所用植物材料:烟草组培苗品种为巴斯玛14号。
一种利用烟草表达三七皂苷的方法,具体包括以下步骤:
1、pCAMBIA2300-35S-SS2-1表达载体构建
1.1SS基因克隆:参照三七基因组中的SS基因序列,在两端分别设计正反引物,克隆三七中的SS基因,序列如SEQ ID NO:1所示;
所述正反引物分别是SS FP:5’-ACGCGTCGACATGGGAAGTTTGGGGGCAATTC-3’,SS RP:5’-TGGTTCTGCAGTCACTGTTTGTTCGGTAGTAG-3’。
1.2SS基因构建到表达载体并转化农杆菌通过SDS碱裂解法提取SS质粒,根据表达载体的切割位点选择EcoRI和HindIII对质粒和载体pCAMBIA2300-35S进行酶切后跑胶检测,正确条带进行切胶回收,回收得到目的基因和载体用T4DNALigase进行连接,连接成功的植物表达载体命名为
pCAMBIA2300-35S-SS2-1,将其转化到农杆菌EHA105中,28℃,180r/min摇菌之后用涂布器将菌液涂布在含卡那霉素的平板上,倒置培养,长出菌落后,挑取若干单克隆作为模板,应用菌落PCR技术,验证转化结果,同时摇菌提取。
2、农杆菌介导SS基因转化烟草在超净工作台将烟叶剪成0.5cm2大小,放入农杆菌浸染液中浸泡3min,取出烟叶,用滤纸吸取多余浸染液后,将叶片转入含有0.5mg/L浓度的6-BA培养基上,黑暗培养3天;经过黑暗培养的烟叶转入含有为500mg/L羧苄青霉素的MS培养基上,25℃±1℃,光照培养30至45天;形成愈伤组织后,嫩芽陆续从中长出,将组织整体转入含有羧苄青霉素和浓度为50mg/L卡那霉素的MS选择压培养基上,25℃±1℃,光照培养7至10天;经过筛选后,继续生长的烟芽从愈伤组织上分离下来,转至生根培养基中,25℃±1℃,光照培养;生根的烟草苗长至4-5cm高,有3到4片小叶时就可炼苗移栽;进一步用CTAB法提取烟草基因组DNA,以转基因烟草苗的DNA做为模板,野生型烟草DNA作为阴性对照,进行PCR扩增目的基因SS。
3、烟草叶片中单体皂苷含量检测:取新鲜烟草叶片,将其表面清洗干净;把叶片放入信封置于40℃烘箱中,将其烘干;用研钵将烘干的叶片研磨成细粉;称取200mg粉末放入50mL离心管中,并加入15mL 70%甲醇,常温超声30min;用5mL注射器吸取大约2mL的甲醇提取试剂过孔径为0.2μm的滤膜,过滤2次;将2次滤液放于棕色样品瓶中,上高效液相色谱仪检测样品中单体皂苷Rb1,Rd,R1,Re,Rg1的含量。
pCAMBIA2300-35S-SS2-1表达载体构建
成功从三七基因组中克隆出SS基因,并构建到表达载体pCAMBIA2300S中(图1)。将重组质粒,导入农杆菌感受态细胞中,摇菌后涂布在含卡那霉素的LB培养基上筛选。菌落长出之后,挑取阳性克隆作为PCR检测的模板扩增,验证是否成功转化,通过跑胶检测到在约1248bp的区域出现条带,与目的基因片段大小一致。同时根据表达载体的切割位点选择EcoRI和HindIII对pCAMBIA2300-35S-SS2-1质粒再次进行双酶切,反向验证连接结果(图2)。结果证明目的基因SS已经成功插入到新载体中,将该植物表达载体命名为pCAMBIA2300-35S-SS2-1。
转SS基因阳性烟草植株
本发明实施例采取经典的农杆菌介导法,取长至4-5片叶期的烟草无菌苗,剪取烟叶经过共培养、除菌培养、筛选培养、生根培养、炼苗移栽;实验共处理264片烟草叶片,通过转入3种选择培养基中进行筛选后,仅有少量的烟草叶片能形成再生烟芽(图3)。在整个筛选的过程中,把阳性烟芽分离进行多次选择培养,假阳性烟苗大量白化死亡。之后对仍存活的烟苗进行练苗移栽(图4),剪取叶片提取DNA,进行PCR检测是否插入目的基因。CTAB法提取烟草基因组DNA,以转基因烟草苗的DNA做为模板,野生型烟草DNA作为阴性对照,进行PCR扩增。PCR结果显示阴性对照和野生型烟草基因组DNA中未扩增出目的基因SS,而选择培养基初步筛选出的阳性烟草基因组DNA成功扩增出SS基因,说明SS基因已经成功整合到烟草基因组中,实验获得了转基因阳性烟草植株(图5)。
转基因烟草中皂苷含量检测
当转基因烟草生长一段时间后采集叶片,用甲醇提取烟叶中的皂苷,上高效液相色谱仪对烟草叶片中单体皂苷Rb1,Rd,R1,Re,Rg1含量进行检测;结果显示,野生型烟草总未检测出皂苷(图6),而在转基因烟草植株中检测到Rg1,Re,Rb1共3种单体皂苷(图7)。
每亩烟草产烟叶100公斤左右,在转基因烟草植株中,1g烟草叶片中Re,Rg1,Rb1单体皂苷含量分别为0.35625mg/g、0.13965mg/g、0.387375mg/g;1g的Re,Rg1及Rb1单体皂苷售价均为:1.6万,每亩烟草Re单体皂苷可以产生57.008万元的收益,Rg1单体皂苷可以产生22.352万元的收益,Rb1单体皂苷可以产生61.984万元的收益。
序列表
<110> 杜云龙
<120> 一种利用烟草表达三七皂苷的方法
<130> 2021.01.22
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1248
<212> DNA
<213> 三七皂苷(squalene synthase,SS)
<400> 1
atgggaagtt tgggggcaat tctgaagcat ccggacgatt tctatccgtt attgaagctt 60
aaatttgcgg ctaggcatgc ggaaaagcag atccctccgg aaccacactg ggccttctgt 120
tactctatgc ttcataaggt ttctcgaagt tttggcctcg tcattcaaca gctcggccct 180
cagctccgcg atgctgtatg cattttttat ttggttcttc gagcacttga cactgttgag 240
gatgacacaa gtatacctac agaggttaaa gtacctatct tgatggcttt tcatcgccac 300
atatatgata aggactggca cttttcatgt ggtacgaagg aatacaaagt tctcatggac 360
gagtttcatc atgtttctaa tgcttttctg gagcttggaa acggttacca ggaggcaata 420
gaagatatta ccatgagaat gggtgcagga atggcaaaat ttatatgcaa ggaggtggag 480
acaatagatg attatgatga atattgtcac tatgtagcag gacttgttgg attagggttg 540
tcaaagctct tccatgcctc tggggcagaa gatttggcta cagattctct gtccaattca 600
atgggtttat ttctccagaa gacaaacata attcgagatt acttggagga cataaatgag 660
ataccaaagt cacgcatgtt ttggcctcgc cagatttgga gtaaatatgt cgataaactt 720
gaggacttaa aatatgagga aaactcagcc aaggcagtgc ggtgcctaaa tgacatggtc 780
acaaatgctt tggttcatgc tgaagattgc ctaaagtaca tgtctgactt gcgagatcct 840
gctatcttcc ggttctgtgc aataccacag attatggcaa ttggaacact agctttatgc 900
ttcaacaaca ctcaagtctt cagaggggta gtgaaaatga gacgtggtct tactgctaaa 960
gttatagacc gaacaaaaac aatgtcagat gtatatggtg ctttcttcga tttttcttgt 1020
ttgctgaagt ccaaggttga caacaatgat cccaatgcta caaaaacttt gagcaggcta 1080
gaagcaattc agaaaacatg caaggagtct ggaaccctgt ccaaaaggaa atcatacata 1140
atcgagagcg agtcaggaca caattcagcc ctgattgcta ttatcttcat tatactagct 1200
atcctttatg catatctatc ttcaaaccta ctaccgaaca aacagtga 1248
Claims (5)
1.一种利用烟草表达三七皂苷的方法,其特征在于,具体包括以下步骤:
(1)参照三七的基因组序列,在两端分别设计正反引物,克隆三七中的SS基因序列如SEQ ID NO:1;
所述正反引物分别是SS FP:5’-ACGCGTCGACATGGGAAGTTTGGGGGCAATTC-3’,SS RP:5’-TGGTTCTGCAGTCACTGTTTGTTCGGTAGTAG-3’;
(2)将三七SS基因克隆到植物表达载体中,使三七SS基因在35S启动子及其它启动子的作用下进行基因转录与表达;
(3)利用三七完整或部分的SS基因转化烟草,使三七SS基因在烟草包括烟草中表达进行皂苷合成。
2.根据权利要求1所述利用烟草表达三七皂苷的方法,其特征在于:步骤(2)中所述所述植物表达载体为pCAMBIA2300。
3.根据权利要求2所述利用烟草表达三七皂苷的方法,其特征在于:将SS基因构建到植物表达载体pCAMBIA2300-35S中,并命名为pCAMBIA2300-35S-SS2-1,将其转化到农杆菌EHA105中,利用农杆菌介导的方法把含SS基因的质粒转化烟草。
4.根据权利要求2或3所述利用烟草表达三七皂苷的方法,其特征在于:表达载体pCAMBIA2300-35S的构建方法为:先将35S-GFP-terminator插入pCAMBIA2300载体上,插入多克隆位点5’EcoRI/HindIII 3’之间;构建pCAMBIA2300-35S-GFP载体,再用5’SalI--PstI3’切开GFP部分,插入SS基因替换原载体的GFP基因。
5.根据权利要求3所述利用烟草表达三七皂苷的方法,其特征在于:步骤(2)利用农杆菌介导的方法把含SS基因的质粒转化烟草的具体过程为:在超净工作台将烟叶剪成0.5cm2大小,放入农杆菌浸染液中浸泡3min,取出烟叶,用滤纸吸取多余浸染液后,将叶片转入含有0.5mg/L浓度的6-BA培养基上,黑暗培养3天;经过黑暗培养的烟叶转入含有为500mg/L羧苄青霉素的MS培养基上,25℃±1℃,光照培养30至45天;形成愈伤组织后,嫩芽陆续从中长出,将组织整体转入含有羧苄青霉素和浓度为50mg/L卡那霉素的MS选择压培养基上,25℃±1℃,光照培养7至10天;经过筛选后,继续生长的烟芽从愈伤组织上分离下来,转至生根培养基中,25℃±1℃,光照培养;生根的烟草苗长至4-5cm高,有3到4片小叶时就可炼苗移栽。
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