CN114591968A - 烟草NtSCL32基因在植物分枝调控中应用 - Google Patents
烟草NtSCL32基因在植物分枝调控中应用 Download PDFInfo
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- CN114591968A CN114591968A CN202210206739.2A CN202210206739A CN114591968A CN 114591968 A CN114591968 A CN 114591968A CN 202210206739 A CN202210206739 A CN 202210206739A CN 114591968 A CN114591968 A CN 114591968A
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Abstract
本发明属于植物基因工程技术领域,具体涉及一个烟草NtSCL32基因在植物分枝调控中应用专利申请事宜。烟草NtSCL32基因与烟草腋芽发育和侧枝形成相关,用于植物株型调控;所述烟草NtSCL32基因,其编码碱基序列长度为1113bp,序列如SEQ ID No.1所示。本申请中,发明人选择烟草NtSCL32基因作为研究对象,通过对该基因沉默后植株表型研究后发现,降低NtSCL32基因表达量后,烟草腋芽增多,分枝明显增加,即,通过降低NtSCL32基因表达量方式可以实现对植物株型调控。基于这一结果,可为针对性培育合适植株株型新品种奠定一定理论基础和技术基础。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及一个烟草NtSCL32基因在植物分枝调控中应用专利申请事宜。
背景技术
植物的分枝发育起始于腋分生组织,植物分枝情况在植物株型调控中具有决定性作用,而植物株型又与作物产量和品质密切相关,因此,植物株型调控研究具有重要的生产应用意义。而已有研究表明,植物侧枝发育虽然受植物激素、环境等多种因素的影响,但归根结底而言,遗传因素对于植物分枝发育仍然是具有基础性决定作用的。
相关植物基因研究工作表明,大量的基因均参与了植物的分枝发育调控过程,比如植物腋分生组织发育起始相关的拟南芥LS基因,它在拟南芥的营养生长阶段通过REVOLUTA(REV)的表达来影响分生组织标志基因SHOOT MERISTEMLESS(STM)的表达,从而影响AM的形成。另有研究表明,水稻中的LAX基因编码bHLH转录因子,它和SPA在水稻分蘖调控中具有冗余性,1ax突变体的小穗完全或部分被抑制。再如,研究表明,TB1(TEOSINTEBRANCHED1)基因是调控玉米腋芽休眠的关键基因,在调控腋芽发育中具有关键作用。
烟草作为一种以收获叶片为生产原料的经济作物,其分枝发育相关基因研究,对于于改良烟草株型和提升烟叶产量,显然具有重要的技术意义。
发明内容
基于株型调控对于烟草生产的重要技术意义,本申请通过对烟草NtSCL32基因与植株分枝影响相关研究,从而为烟草株型调控奠定一定技术基础。
本申请所采取的技术方案简述如下。
烟草NtSCL32基因在植物分枝调控中应用,烟草NtSCL32基因与烟草腋芽发育和侧枝形成相关,可用于植物株型调控;所述植物,具体例如为烟草;
具体应用时,通过基因工程技术手段,通过将NtSCL32基因沉默进而抑制NtSCL32基因表达翻译为NtSCL32蛋白后,基因沉默植株中,烟草腋芽生长数量明显增加、侧枝明显增多(即,通过基因沉默方式促进烟草腋芽增多、促进侧枝生长);
所述烟草NtSCL32基因,其编码碱基序列长度为1113bp,序列如SEQ ID No.1所示;具体如下:
ATGTGGGTACTCAACAATTTGGCTTCTTCTAACGGAGATCCAAATCAAAGGCTTACATCTTGGTTTCTTAGGGCATTAATCTCAAGGGCTTCTAGGGTTTGTCCTACAGCCACAAATAATTATCTCTATGGAAGTAGTAATCTTGATCAAAGGAGATTAATGAGTGTGACTGAGCTTGCAGGGTATGTAGATCTCATCCCTTGGCATAGATTTGGATTTTGTGTATCAAATAGTGCTATTTATAAGGCCATTGAAGGGCACACAAAAGTTCACATATTAGATTTTAGTATCACTCATTGTATGCAATGGCCAACTCTAATTGATGCAATAGCTAAGAGGCCTGAGGGTCCTCCTTCTCTTCGAATATCGGTGCCTTCTTGGAGGCCACCAGTTCCTCCATTGCTTAATGTATCAAGTGAAGAAGTTGGCCATCGTTTGGCTAATTTTGCAAAGTTTAGAGATGTCCCTTTTGAATTCCAAGTGATTGAAGACTTAAACTATGATATGCTCTTGAGCCAATTGAGTCCTTTAACTCTTCAACTTAGGGATGATGAGGCTTTGGTGGTTAATTGCCAAAATTGGTTAAGGTATATGCCCGATGAACAAATTAATGGAAGTAGTTATTCTTCTCGTGACATATTTCTTGATATGGTTAAGGAACTAAATCCATGTATTATGACTGTGGTTGATGAGGATTCTGACTTGGGAAACTCAAGTTTAACATCAAGAATAGCCACTTGCTTTAATTATTTATGGATACCCTTTGATGCATTAGAGACTTTCTTGCCTAAGGATAGTAGACAAAGGCTTGATTATGAAGCTGATATTGGCCACAAAATTGAAAACATTATTGGAGTTGAAGGGGTTCAAAGGATAGAGAGATTAGAGTGTTGCAATAAATTCTCACAAAGGATGGAAAATAGTGGTTTTATGAGTGTTCCTTTTTGTGAGGAAACAATTAAGGAAGTGAAGTCACTGTTAGATGAGCGTGCAAGTGGGTGGGGTATGAAAAAAGAGGATGACATGCTTGTATTGACATGGAAAGGCCATAACTCTGTCTATGCAACTGCTTGGATCTTGGTCCCGCCTCAAATGGACATGACGATTGAGTAA;
烟草NtSCL32基因所编码烟草分枝调控蛋白NtSCL32的氨基酸序列长度为370AA,氨基酸序列如SEQ ID No.2所示,具体如下:
MWVLNNLASSNGDPNQRLTSWFLRALISRASRVCPTATNNYLYGSSNLDQRRLMSVTELAGYVDLIPWHRFGFCVSNSAIYKAIEGHTKVHILDFSITHCMQWPTLIDAIAKRPEGPPSLRISVPSWRPPVPPLLNVSSEEVGHRLANFAKFRDVPFEFQVIEDLNYDMLLSQLSPLTLQLRDDEALVVNCQNWLRYMPDEQINGSSYSSRDIFLDMVKELNPCIMTVVDEDSDLGNSSLTSRIATCFNYLWIPFDALETFLPKDSRQRLDYEADIGHKIENIIGVEGVQRIERLECCNKFSQRMENSGFMSVPFCEETIKEVKSLLDERASGWGMKKEDDMLVLTWKGHNSVYATAWILVPPQMDMTIE。
烟草NtSCL32基因PCR扩增用引物序列,具体设计如下:
NtSCL32-F: 5'-ATGTGGGTACTCAACAATTTGGCTT-3',
NtSCL32-R: 5'-TTACTCAATCGTCATGTCCATTTGA-3'。
用于敲低烟草NtSCL32基因表达的重组载体,该重组载体命名为pCambia2301-RTM-NtSCL32,通过如下步骤构建获得:
(一)设计引物,并进行PCR扩增
分别设计反、正向片段扩增引物:
NtSCL32-R(+):5'-AGCCTGCAGCCATGGTGAAGGAGGACCCTCAGGCCTCTTA-3',
NtSCL32-R(-):5'-ATAGACTTACAACGTTCAAGGGCTTCTAGGGTTTGTCCTA-3';
利用上述引物扩增反向片段序列如下:
GAGGACCCTCAGGCCTCTTAGCTATTGCATCAATTAGAGTTGGCCATTGCATACAATGAGTGATACTAAAATCTAATATGTGAACTTTTGTGTGCCCTTCAATGGCCTTATAAATAGCACTATTTGATACACAAAATCCAAATCTATGCCAAGGGATGAGATCTACATACCCTGCAAGCTCAGTCACACTCATTAATCTCCTTTGATCAAGATTACTACTTCCATAGAGATAATTATTTGTGGCTGTAGGACAAACCCTAGAAGCCC;
NtSCL32-F(+):5'-GCGGGTCGACGGTACCTCAAGGGCTTCTAGGGTTTGTCCTA-3',
NtSCL32-F(-):5'-GACCCAGCAGATAGATGAAGGAGGACCCTCAGGCCTCTTA-3';
利用上述扩增正向片段序列如下:
GGGCTTCTAGGGTTTGTCCTACAGCCACAAATAATTATCTCTATGGAAGTAGTAATCTTGATCAAAGGAGATTAATGAGTGTGACTGAGCTTGCAGGGTATGTAGATCTCATCCCTTGGCATAGATTTGGATTTTGTGTATCAAATAGTGCTATTTATAAGGCCATTGAAGGGCACACAAAAGTTCACATATTAGATTTTAGTATCACTCATTGTATGCAATGGCCAACTCTAATTGATGCAATAGCTAAGAGGCCTGAGGGTCCTC
随后,以烟草cDNA为模板,分别利用上述引物进行PCR扩增,并对扩增产物进行提取、纯化备用,获得干扰载体的目标序列片段;
(二)酶切、连接
对pCambia2301-RTM载体进行BamHI和XbaI双酶切,并利用T4_ligase将酶切产物与步骤(一)中的所回收反向片段的PCR扩增产物进行连接;
(三)转化和筛选
将步骤(二)中反向片段连接产物转化大肠杆菌感受态细胞,筛选、鉴定获得重组正确的中间质粒载体;
对中间载体质粒进行KpnI单酶切后,与步骤(一)中的正向片段扩增产物机械能重组反应;
进一步,将重组反应产物转化大肠杆菌感受态细胞,并最终筛选、鉴定获得重组正确pCambia2301-RTM-NtSCL32。
所述重组载体pCambia2301-RTM-NtSCL32在植物中应用,将该重组载体转化植物后,能够降低NtSCL32基因翻译表达水平,进而促进植物腋芽发育和侧枝生长,从而实现株型调控效果。
一种调控植物株型的植物新品种培育方法,利用农杆菌介导的基因转化方法,将重组载体pCambia2301-RTM-NtSCL32转化植物后,进一步筛选、鉴定获得NtSCL32基因表达降低新品种,该新品种腋芽生长发育较多、较快,且侧枝形成较多。
基于前期工作积累,本申请中,发明人选择烟草NtSCL32基因作为研究对象,通过对该基因沉默后植株表型研究后发现,降低NtSCL32基因表达量后,烟草腋芽增多,分枝明显增加,即,通过降低NtSCL32基因表达量方式可以实现对植物株型调控。基于这一结果,可为针对性培育合适植株株型新品种奠定一定理论基础和技术基础。
附图说明
图1为NtSCL32克隆的PCR电泳图;
图2为干扰植株中NtSCL32基因表达量分析结果;图中,L-10、 L-12、L-15、L-7表示不同转基因株系;
图3为NtSCL32干扰植株烟草分枝表型。
具体实施方式
下面结合实施例对本申请做进一步的解释说明。在介绍具体实施例前,就下述实施例中部分实验背景情况简要介绍说明如下。
烟草品种:K326,一种常见的栽培烟草品种,实施例中所采用种子由国家烟草基因研究中心保存提供;
载体:pEASY-T1 Simple 载体,购自北京全式金生物技术有限公司;
pCambia2301-RTM干扰载体,由上海凯益生物科技有限公司提供。
菌株:
Trans1-T1化学感受态细胞,购自北京全式金生物技术有限公司;
LBA4404农杆菌菌株,生物实验中常用菌株,可公开获得;
引物的合成和DNA测序由北京六合华大基因科技股份有限公司提供完成;
实验试剂:
荧光定量PCR酶(SYBR qPCR kit),购自郑州安赛生物科技有限公司;
反转录试剂盒、T4连接酶、限制性内切酶,购自宝生物工程(大连)有限公司;
PCR 扩增酶,2×TransStart GoldPfu PCR SuperMix,荧光定量PCR酶、DNA扩增酶,购自北京全式金生物技术有限公司;
RNA 提取试剂盒(SuperPure Plant polyRNA Kit)、DNA纯化胶回收试剂盒购自QIAGEN公司。
实施例1
基于前期研究工作总结,发明人选择烟草NtSCL32基因作为研究对象。为便于对该基因对植物生长实际影响情况进行明确,发明人首先对该基因进行了克隆。具体过程简介如下。
(1)制备cDNA作为克隆模板
取旺长期烟草(K326)的根(基于该基因的组织表达模式分析结果,该基因主要在根部表达,在叶片中表达量很低)100mg作为样品,在液氮中充分研磨,参照RNA提取试剂盒说明书提取总RNA,然后反转录为cDNA备用;
(2)设计引物,进行PCR扩增
设计用于扩增NtSCL32基因的引物序列如下:
NtSCL32-F: 5'-ATGTGGGTACTCAACAATTTGGCTT-3',
NtSCL32-R: 5'-TTACTCAATCGTCATGTCCATTTGA-3';
以步骤(1)中所制备cDNA为模板,利用上述引物进行PCR扩增,
50 μl扩增体系设计为:
cDNA模板,2 μl;
上、下游引物各1 ul;
2×TransStart GoldPfu PCR SuperMix,25μl;
ddH2O加至50 ul;
PCR扩增程序为:94℃预变性4min;94℃变性30s,58℃退火30s,72℃延伸1min,共30个循环;再72℃终延伸10min。
对PCR扩增产物进行电泳检测分析后,参照胶回收试剂盒说明书,对PCR扩增产物进行回收纯化。
(3)与pEASY-T1载体连接、转化
将步骤(2)中所提取PCR扩增产物连接到pEASY-T1载体上,具体连接体系参考如下:
PCR扩增产物,6μL;
pEASY-T1 载体,1μL;
混匀后,25℃连接25 min。
随后,将上述连接产物转化至大肠杆菌感受态细胞中,具体转化过程操作参考如下:
冰上溶解感受态细胞后,加连接产物于50μL的Trans1-T1感受态细胞中,轻弹混匀,冰浴30 min;42℃水浴中热激30s,立即置于冰上2min;加入250μL平衡至室温的LB(不含抗生素),37℃摇荡培养1h;混合后均匀地涂布到LB固体平板(含60μg/μL 氨苄青霉素)平板上,倒置培养皿,37℃培养过夜。
挑取白斑扩增培养后,提取质粒DNA,并对重组质粒进行菌液PCR鉴定(结果如图1所示),随后,将重组正确的阳性重组质粒送样测序,获得NtSCL32基因序列。
测序分析结果表明,NtSCL32基因序列具体如SEQ ID NO.1所示,其所编码的NtSCL32蛋白的氨基酸序列如SEQ ID NO.2所示。进一步结构分析组织表达模式分析表明,该基因位于烟草7号染色体上,为单外显子结构,在根烟草根系中表达较高。
实施例2
为进一步了解NtSCL32基因在植株生长发育中的作用,发明人构建了用于敲低NtSCL32基因的重组表达载体RNAi-NtSCL32,下面就该重组载体的构建过程简要介绍如下。
(一)设计引物,并进行PCR扩增
设计反向片段扩增引物:
NtSCL32-R(+):5'-AGCCTGCAGCCATGGTGAAGGAGGACCCTCAGGCCTCTTA-3',
NtSCL32-R(-):5'-ATAGACTTACAACGTTCAAGGGCTTCTAGGGTTTGTCCTA-3';
所扩增反向片段目的序列如下:
GAGGACCCTCAGGCCTCTTAGCTATTGCATCAATTAGAGTTGGCCATTGCATACAATGAGTGATACTAAAATCTAATATGTGAACTTTTGTGTGCCCTTCAATGGCCTTATAAATAGCACTATTTGATACACAAAATCCAAATCTATGCCAAGGGATGAGATCTACATACCCTGCAAGCTCAGTCACACTCATTAATCTCCTTTGATCAAGATTACTACTTCCATAGAGATAATTATTTGTGGCTGTAGGACAAACCCTAGAAGCCC;
设计正向片段扩增引物:
NtSCL32-F(+):5'-GCGGGTCGACGGTACCTCAAGGGCTTCTAGGGTTTGTCCTA-3',
NtSCL32-F(-):5'-GACCCAGCAGATAGATGAAGGAGGACCCTCAGGCCTCTTA-3';
所扩增正向片段目的序列如下:
GGGCTTCTAGGGTTTGTCCTACAGCCACAAATAATTATCTCTATGGAAGTAGTAATCTTGATCAAAGGAGATTAATGAGTGTGACTGAGCTTGCAGGGTATGTAGATCTCATCCCTTGGCATAGATTTGGATTTTGTGTATCAAATAGTGCTATTTATAAGGCCATTGAAGGGCACACAAAAGTTCACATATTAGATTTTAGTATCACTCATTGTATGCAATGGCCAACTCTAATTGATGCAATAGCTAAGAGGCCTGAGGGTCCTC。
随后,分以烟草cDNA为模板,分别利用上述引物进行PCR扩增,并对扩增产物进行提取、纯化备用,获得干扰载体的目标序列片段;
(二)酶切、连接
对pCambia2301-RTM载体进行BamHI和XbaI双酶切,并利用T4_ligase将酶切产物与步骤(一)中的所回收反向片段的PCR扩增产物进行连接;
(三)转化和筛选
将步骤(二)中反向片段连接产物转化大肠杆菌感受态细胞,筛选、鉴定获得重组正确的中间质粒载体;
对中间载体质粒进行KpnI单酶切后,与步骤(一)中的正向片段扩增产物进行重组反应(相关操作参考现有技术常规操作即可,不再赘述);
进一步,将重组反应产物转化大肠杆菌感受态细胞,并最终筛选、鉴定获得重组正确pCambia2301-RTM-NtSCL32。
实施例3
基于农杆菌介导的转化法,发明人进一步将实施例2所构建重组载体pCambia2301-RTM-NtSCL32转化了烟草植株,以期获得NtSCL32基因表达量降低或者NtSCL32基因敲除的转基因植株新品种。具体实验过程简要介绍如下。
(1)转化农杆菌
在冰上冻融农杆菌感受态细胞后,加入6 μL实施例3所制备载体pCambia2301-RTM-NtSCL32,轻弹混匀;随后将混合物置于预冷的电转杯中,冰上放置5 min;
将电转仪参数调至:电压2.5 kV,电容25 μF,电阻200 Ω;然后用吸水纸将电转杯外壁上的水滴吸除干净后,把电转杯放入电击槽中,电击5 ms;
迅速加入800 μL的预热至28℃的YEB液体培养基,220 rpm、28℃振荡复苏3 h;
然后将菌液4500 rpm离心1 min,弃一半体积的上清,重新悬浮后均匀涂于含有Rif(100 μg/mL)、Str(50 μg/mL)和Kan(50 μg/mL)的YEB固体培养基上,28℃倒置培养约2~3 d,直至单菌落形成;
挑取单菌落,扩培后对菌液进行PCR鉴定,鉴定正确的阳性克隆菌株,即为转化正确的农杆菌工程菌。
进一步将农杆菌工程菌培养至OD600=0.6左右,4000 rpm离心5 min收集菌体,再用20 mL的MS液体培养基悬浮菌体,以此作为后续转染用侵染液。
(2)转化烟草植株
取生长一个月左右的K326烟草无菌苗叶片,用打孔器将叶片处理成直径0.5 cm大小的叶盘,将处理后叶盘在MS固体培养基上预培养3 d;
随后,将上述预培养后叶盘置于步骤(1)中的侵染液中,充分侵染10 min;
用无菌的滤纸吸干浸染后叶盘周围多余的菌液,再在MS+6-BA(2 mg/L)+NAA(0.5mg/L)的固体培养基上暗培养3 d;
用含有Cef(400 mg/L)的无菌水清洗叶盘,并用无菌滤纸吸去多余的液体,将叶盘转到含有6-BA(2 mg/L)、NAA(0.5 mg/L)、Cef(200 mg/L)和Kan(50 mg/L)的MS固体筛选培养基上,28℃光照培养;
待不定芽长到0.5cm时,转移到含Cef(200 mg/L)和Kan(50 mg/L)的MS固体培养基上生根。
待生长一个月左右,取少量叶片,提取DNA,并通过PCR方法检测阳性转基因株系。具体PCR鉴定时,引物设计为:
NtSCL32-J-F: 5'-GACGCACAATCCCACTATCC-3',
NtSCL32-J-R: 5'-TGAAGGAGGACCCTCAGGCCTCTTA-3'。
转基因株系表型变化情况:
进一步地,基于实时定量PCR技术,对鉴定阳性转基因株系中NtSCL32基因表达情况进行检测分析(具体操作参考前述及现有技术即可)。分析可以看出,与野生型K326相比,在不同RNAi植株株系中,烟草NtSCL32基因表达水平均明显降低(图2),也即,表明成功构建获得了NtSCL32基因表达量降低的转基因植株新品种。
进一步T0代植株移栽至盆中后,温室培养至6周时,对阳性株的腋芽长度表型进行观察,结果如图3所示。可以看出,相较于野生型K326,转基因株系腋芽数量明显增多、生长速度较快,侧枝分枝数量也明显增多。基于此结果,可有效调控相关植物尤其是烟草株型。
SEQUENCE LISTING
<110> 中国烟草总公司郑州烟草研究院
<120> 烟草NtSCL32基因在植物分枝调控中应用
<130> none
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1113
<212> DNA
<213> Nicotiana tabacum
<400> 1
atgtgggtac tcaacaattt ggcttcttct aacggagatc caaatcaaag gcttacatct 60
tggtttctta gggcattaat ctcaagggct tctagggttt gtcctacagc cacaaataat 120
tatctctatg gaagtagtaa tcttgatcaa aggagattaa tgagtgtgac tgagcttgca 180
gggtatgtag atctcatccc ttggcataga tttggatttt gtgtatcaaa tagtgctatt 240
tataaggcca ttgaagggca cacaaaagtt cacatattag attttagtat cactcattgt 300
atgcaatggc caactctaat tgatgcaata gctaagaggc ctgagggtcc tccttctctt 360
cgaatatcgg tgccttcttg gaggccacca gttcctccat tgcttaatgt atcaagtgaa 420
gaagttggcc atcgtttggc taattttgca aagtttagag atgtcccttt tgaattccaa 480
gtgattgaag acttaaacta tgatatgctc ttgagccaat tgagtccttt aactcttcaa 540
cttagggatg atgaggcttt ggtggttaat tgccaaaatt ggttaaggta tatgcccgat 600
gaacaaatta atggaagtag ttattcttct cgtgacatat ttcttgatat ggttaaggaa 660
ctaaatccat gtattatgac tgtggttgat gaggattctg acttgggaaa ctcaagttta 720
acatcaagaa tagccacttg ctttaattat ttatggatac cctttgatgc attagagact 780
ttcttgccta aggatagtag acaaaggctt gattatgaag ctgatattgg ccacaaaatt 840
gaaaacatta ttggagttga aggggttcaa aggatagaga gattagagtg ttgcaataaa 900
ttctcacaaa ggatggaaaa tagtggtttt atgagtgttc ctttttgtga ggaaacaatt 960
aaggaagtga agtcactgtt agatgagcgt gcaagtgggt ggggtatgaa aaaagaggat 1020
gacatgcttg tattgacatg gaaaggccat aactctgtct atgcaactgc ttggatcttg 1080
gtcccgcctc aaatggacat gacgattgag taa 1113
<210> 2
<211> 370
<212> PRT
<213> Nicotiana tabacum
<400> 2
Met Trp Val Leu Asn Asn Leu Ala Ser Ser Asn Gly Asp Pro Asn Gln
1 5 10 15
Arg Leu Thr Ser Trp Phe Leu Arg Ala Leu Ile Ser Arg Ala Ser Arg
20 25 30
Val Cys Pro Thr Ala Thr Asn Asn Tyr Leu Tyr Gly Ser Ser Asn Leu
35 40 45
Asp Gln Arg Arg Leu Met Ser Val Thr Glu Leu Ala Gly Tyr Val Asp
50 55 60
Leu Ile Pro Trp His Arg Phe Gly Phe Cys Val Ser Asn Ser Ala Ile
65 70 75 80
Tyr Lys Ala Ile Glu Gly His Thr Lys Val His Ile Leu Asp Phe Ser
85 90 95
Ile Thr His Cys Met Gln Trp Pro Thr Leu Ile Asp Ala Ile Ala Lys
100 105 110
Arg Pro Glu Gly Pro Pro Ser Leu Arg Ile Ser Val Pro Ser Trp Arg
115 120 125
Pro Pro Val Pro Pro Leu Leu Asn Val Ser Ser Glu Glu Val Gly His
130 135 140
Arg Leu Ala Asn Phe Ala Lys Phe Arg Asp Val Pro Phe Glu Phe Gln
145 150 155 160
Val Ile Glu Asp Leu Asn Tyr Asp Met Leu Leu Ser Gln Leu Ser Pro
165 170 175
Leu Thr Leu Gln Leu Arg Asp Asp Glu Ala Leu Val Val Asn Cys Gln
180 185 190
Asn Trp Leu Arg Tyr Met Pro Asp Glu Gln Ile Asn Gly Ser Ser Tyr
195 200 205
Ser Ser Arg Asp Ile Phe Leu Asp Met Val Lys Glu Leu Asn Pro Cys
210 215 220
Ile Met Thr Val Val Asp Glu Asp Ser Asp Leu Gly Asn Ser Ser Leu
225 230 235 240
Thr Ser Arg Ile Ala Thr Cys Phe Asn Tyr Leu Trp Ile Pro Phe Asp
245 250 255
Ala Leu Glu Thr Phe Leu Pro Lys Asp Ser Arg Gln Arg Leu Asp Tyr
260 265 270
Glu Ala Asp Ile Gly His Lys Ile Glu Asn Ile Ile Gly Val Glu Gly
275 280 285
Val Gln Arg Ile Glu Arg Leu Glu Cys Cys Asn Lys Phe Ser Gln Arg
290 295 300
Met Glu Asn Ser Gly Phe Met Ser Val Pro Phe Cys Glu Glu Thr Ile
305 310 315 320
Lys Glu Val Lys Ser Leu Leu Asp Glu Arg Ala Ser Gly Trp Gly Met
325 330 335
Lys Lys Glu Asp Asp Met Leu Val Leu Thr Trp Lys Gly His Asn Ser
340 345 350
Val Tyr Ala Thr Ala Trp Ile Leu Val Pro Pro Gln Met Asp Met Thr
355 360 365
Ile Glu
370
Claims (6)
1.烟草NtSCL32基因在植物分枝调控中应用,其特征在于,烟草NtSCL32基因与烟草腋芽发育和侧枝形成相关,用于植物株型调控;
所述烟草NtSCL32基因,其编码碱基序列长度为1113bp,序列如SEQ ID No.1所示。
2.如权利要求1所述烟草NtSCL32基因在植物分枝调控中应用,其特征在于,所述植物,具体为烟草;
具体应用时,通过基因工程技术手段,通过将NtSCL32基因沉默进而抑制NtSCL32基因表达翻译为NtSCL32蛋白后,基因沉默植株中,烟草腋芽生长数量明显增加、侧枝明显增多。
3.烟草NtSCL32基因PCR扩增用引物序列,其特征在于,所述烟草NtSCL32基因,,序列如SEQ ID No.1所示,PCR扩增时,具体引物序列设计如下:
NtSCL32-F: 5'-ATGTGGGTACTCAACAATTTGGCTT-3',
NtSCL32-R: 5'-TTACTCAATCGTCATGTCCATTTGA-3'。
4.用于敲低烟草NtSCL32基因表达的重组载体pCambia2301-RTM-NtSCL32,其特征在于,
通过如下步骤构建获得:
(一)设计引物,并进行PCR扩增
分别设计正、反向片段扩增引物:
NtSCL32-R(+):5'-AGCCTGCAGCCATGGTGAAGGAGGACCCTCAGGCCTCTTA-3',
NtSCL32-R(-):5'-ATAGACTTACAACGTTCAAGGGCTTCTAGGGTTTGTCCTA-3';
NtSCL32-F(+):5'-GCGGGTCGACGGTACCTCAAGGGCTTCTAGGGTTTGTCCTA-3',
NtSCL32-F(-):5'-GACCCAGCAGATAGATGAAGGAGGACCCTCAGGCCTCTTA-3';
随后,以烟草cDNA为模板,利用上述引物进行PCR扩增,并对扩增产物进行提取、纯化备用,获得干扰载体的目标序列片段;
(二)酶切、连接
对pCambia2301-RTM载体进行BamHI和XbaI双酶切,并利用T4_ligase将酶切产物与步骤(一)中的所回收反向片段的PCR扩增产物进行连接;
(三)转化和筛选
将步骤(二)中反向片段连接产物转化大肠杆菌感受态细胞,筛选、鉴定获得重组正确的中间质粒载体;
对中间载体质粒进行KpnI单酶切后,与步骤(一)中的正向片段扩增产物进行重组反应;
进一步,将重组反应产物转化大肠杆菌感受态细胞,并最终筛选、鉴定获得重组正确载体pCambia2301-RTM-NtSCL32。
5.权利要求4所述重组载体pCambia2301-RTM-NtSCL32在植物中应用,其特征在于,将该重组载体转化植物后,能够降低NtSCL32基因翻译表达水平,进而促进植物腋芽发育和侧枝生长,从而实现株型调控。
6.一种调控植物株型的植物新品种培育方法,其特征在于,利用农杆菌介导的基因转化方法,将重组载体pCambia2301-RTM-NtSCL32转化植物后,进一步筛选、鉴定获得NtSCL32基因表达降低新品种,该新品种腋芽生长发育较多、较快,且侧枝形成较多。
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