CN113151322B - 一种烟草淀粉合酶基因及其应用 - Google Patents
一种烟草淀粉合酶基因及其应用 Download PDFInfo
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- CN113151322B CN113151322B CN202110574080.1A CN202110574080A CN113151322B CN 113151322 B CN113151322 B CN 113151322B CN 202110574080 A CN202110574080 A CN 202110574080A CN 113151322 B CN113151322 B CN 113151322B
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- C12N9/10—Transferases (2.)
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Abstract
本发明涉及一种烟草淀粉合酶基因及其应用,碱基序列具体如SEQ IDNO.1所示。本申请中,通过对烟草淀粉合酶NtGLGA的初步研究,发现其与烟草叶片中淀粉的合成代谢相关。在本氏烟草中将该基因沉默,烟草叶片中支链淀粉和总淀粉含量明显降低,支链淀粉降低了64.8%,总淀粉含量降低了30.2%。基于烟草淀粉合酶NtGLGA这一特性,可通过基因工程育种为烟叶品质调控、烟草新品种培育提供参考借鉴。
Description
技术领域
本发明属于烟草基因工程技术领域,具体涉及一种烟草淀粉合酶基因及其应用。
背景技术
淀粉是烟叶中最重要的基础有机化合物,成熟的鲜烟叶中淀粉含量高达40%左右,决定着烟叶内在品质和外观商品等级。淀粉含量是评价烟叶质量的关键因素之一。鲜烟淀粉含量高对品质是不利的,尤其对烟叶色泽、香味不利;烤后烟叶尽管淀粉含量较低,但仍会影响烟叶外观及内在品质。以淀粉形态存在的糖类在烟支燃吸时,一方面对烟气质量会产生不良影响,另一方面影响燃烧速度和完全性。另外,淀粉在燃烧时会产生焦糊气味,破坏烟草燃吸时所形成的香味。我国烤烟中淀粉含量(质量分数)约为4%~6%,而国外优质烤烟淀粉含量仅为1%~2%。
因此,烟草中影响淀粉含量的基因功能的研究将为烟叶品质改善、烟草品种遗传改良提供理论支持,对改善我国烟草产品品质具有重要的意义。
发明内容
本发明的目的是提供一种烟草淀粉合酶基因及其应用,以解决现烤烟中淀粉含量过高的问题,从而为烟叶品质调控、烟草新品种培育奠定一定的基础。
为实现上述目的,本发明是通过以下技术方案实现的:
一种烟草淀粉合酶基因,碱基序列具体如SEQ ID NO.1所示,含有3636个碱基,命名为NtGLGA。
进一步的,烟草淀粉合酶基因的氨基酸序列如SEQ ID NO.2所示,由1211个氨基酸残基组成。
进一步的,烟草淀粉合酶基因的PCR扩增制备方法,包括如下步骤:
(1)提取基因组,并反转录为cDNA备用;
(2)设计PCR扩增用引物,并进行PCR扩增,具体引物序列设计如下:
NtGLGA-F:5’-TTGGAGTTTCTTCTGCAAGGTG-3’,
NtGLGA-R:5’-CAATTACAGGGTTTCCAGACAC-3’。
进一步的,在步骤(1)中提取基因组时,以烟草品种红花大金元叶片为样品。
上述任一项的烟草淀粉合酶基因的应用,该基因表达的蛋白与植物叶片中淀粉含量相关,降低该蛋白表达后,叶片中支链淀粉和总淀粉含量明显降低。
进一步的,利用基因沉默技术、或者基因超表达方法,通过调节烟草淀粉合酶表达量,来调节控制烟叶中淀粉含量。
进一步的,通过转基因技术、瞬时表达技术或基因组编辑技术,构建含有烟草淀粉合酶基因的病毒诱导沉默载体、RNAi干涉载体、超表达载体,转化烟草,筛选获得淀粉含量变化的烟草新品种。
具体例如:利用病毒诱导的基因沉默(VIGS)的技术,干扰烟草淀粉合酶基因的表达使其沉默,烟草淀粉合酶基因沉默植株中支链淀粉和总淀粉含量显著下降,进而获得淀粉含量下降的植物新品种。
本发明的有益效果是:
本申请中,通过对特定烟草淀粉合酶基因的初步研究,发现其与烟草叶片淀粉含量高度相关,在本氏烟草中将该基因沉默,叶片中淀粉含量发生了明显降低。基于这一特性,可为烟叶品质调控、烟草新品种培育提供一定的应用基础和参考借鉴。
附图说明
图1为与对照植株相比,烟草淀粉合酶基因沉默植株中该基因的相对表达量;
图2为病毒诱导基因沉默的烟叶及对照烟叶中的淀粉含量比较。
具体实施方式
以下通过实施例来详细说明本发明的技术方案,以下的实施例仅是示例性的,仅能用来解释和说明本发明的技术方案,而不能解释为是对本发明技术方案的限制。
生物材料:
本氏烟草,一种现常用烟草材料,育苗钵中育苗,待发芽后两周进行分苗,种于塑料钵(10cm×10cm)中,22℃、16h光/8h暗条件下进行日常肥水管理等栽培管理。
下述实施例中所采用的VIGS载体是一种来自烟草脆裂病毒的病毒载体(tobaccorattle virus,TRV),所具体利用的TRV2(一种常用载体)带有卡那霉素筛选标记和35S启动子,同时TRV2带有EcoR I和BamH I等多克隆位点,可以用来携带和转化外源基因。
实验试剂:
LB液体培养基,1L含量中含有:10g细菌蛋白胨(bacteriological peptone);10g氯化钠(NaCl);5g酵母抽提物(yeast extract),高温高压灭菌。
YEB液体培养基,1L含量中含有:5g牛肉浸膏(beef extract);5g细菌蛋白胨(bacteriological peptone);5g蔗糖(sucrose);1g酵母抽提物(yeast extract);2mL 1M硫酸镁(MgSO4),高温高压灭菌。
1M 2-(N-吗啉)乙磺酸(MES)储备液:ddH2O溶解,过滤灭菌,-20℃储存备用。
200mM乙酰丁香酮(Acetosyringone,As)储备液:二甲基亚砜(DSMO)溶解,-20℃储存备用。
实施例1
本实施例就烟草NtGLGA基因克隆及沉默载体的构建过程简要介绍如下。
(1)烟草NtGLGA基因克隆
根据前期对于烟草基因组及相关NtGLGA基因研究,选择特异编码序列为目标片段,设计PCR扩增用引物序列如下:
NtGLGA-F:5’-TTGGAGTTTCTTCTGCAAGGTG-3’,
NtGLGA-R:5’-CAATTACAGGGTTTCCAGACAC-3’。
以烟草红花大金元叶片(先提取基因组,再反转录为cDNA)的cDNA为模板,进行PCR扩增获得NtGLGA基因。
PCR扩增程序为:95℃预变性3min;95℃变性15s,53℃退火15s,72℃延伸3min,34个循环后,72℃彻底延伸5min。
对PCR扩增产物进行琼脂糖凝胶电泳检测,并回收电泳产物备用。
(2)构建重组TRV2-NtGLGA载体
将步骤(1)中的PCR扩增产物进行EcoRI、BamHI双酶切,同时对空载体TRV2进行EcoRI、BamHI双酶切,分别回收酶切产物,利用T4 DNA连接酶进行连接。
将连接产物转化大肠杆菌感受态DH5α,转化操作结束后将转化产物涂布在含50mg/L Kan的LB固体培养基上,37℃过培养夜。
挑选阳性单菌落扩增后进一步进行PCR鉴定,并结合测序验证,确保获得构建正确的重组载体TRV2-NtGLGA。
需要说明的是,烟草NtGLGA基因,包括3636个碱基,碱基序列如SEQ ID NO.1所示。
烟草淀粉合酶蛋白NtGLGA,包括1211个氨基酸,氨基酸序列如SEQ ID NO.2所示。
实施例2
在实施例1基础上,利用农杆菌介导的VIGS技术,进一步将所构建的重组TRV2-NtGLGA载体转化了烟草植株,并就相关植物表型变化情况做了验证分析,具体实验过程简介如下。
(1)转化农杆菌
需要说明的是,参考实施例1操作及现有技术,同时制备了TRV2-GFP重组载体作为对照,具体转化过程为:
将TRV2-GFP(载体对照)及TRV2-NtGLGA的阳性克隆质粒,分别通过电击转化方式转化进入农杆菌GV3101感受态细胞中,利用含50mg/L Kan和50mg/L Rif的YEB平板进行培养筛选,在28℃倒置培养2d后,利用菌落PCR筛选带有目的基因的农杆菌。
(2)制备转染用菌液
将步骤(1)中筛选所得阳性农杆菌克隆在5mL的YEB液体培养基(含50mg/L Kan和50mg/L Rif)中,28℃、250rpm条件下培养过夜。
取50uL过夜培养物接种至50mL的YEB液体培养基(含50mg/L Kan)中,培养至OD600=1.0-1.5左右,然后4000g离心5min,收集菌体,再用MMA重悬,调节OD600=1.0左右。
最后室温放置3h左右后,作为转染用菌液。
(3)瞬时转化
以3-4w(周)苗龄的本氏烟草叶片为实验材料,利用1mL规格注射器,将步骤(2)中所制备转染用菌液注射至烟草叶片中,注射后的烟草继续在人工培养箱内培养,观察表型变化。
进一步通过qRT-PCR对NtGLGA基因表达情况进行了检测,结果如图1所示,可以看出,TRV2-NtGLGA的侵染植株中,NtGLGA的表达量显著降低,qRT-PCR引物如下:
NtGLGA-F:5’-ACAGGCCAACCTTCCTTTG-3’,
NtGLGA-R:5’-GCAGAACCAAGCAAGACCA-3’。
进一步地,对实验组(TRV2-NtGLGA浸染植株)和对照组(TRV2-GFP浸染植株)中的叶片淀粉含量情况进行了检测(检测方法参照:格锐思生物的支链-直链-总淀粉含量试剂盒(分光光度法)),结果如图2所示。
从图2结果可以看出,实验组中支链淀粉含量和总淀粉含量与对照组相比均有显著下降,其中支链淀粉下降了64.2%,总淀粉含量下降了30.2%。这进一步表明,通过沉默NtGLGA基因,可对烟草叶片中淀粉的含量进行调控,进而可为烟叶品质调控、烟草新品种培育奠定一定技术基础。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
序列表
<110> 云南中烟工业有限责任公司
<120> 一种烟草淀粉合酶基因及其应用
<130> WPC211438
<141> 2021-05-25
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3636
<212> DNA
<213> 人工序列(NtGLGA)
<400> 1
atggatgttc cattgccact gcaaagacca ttgagttaca gcaatagtgt ctccactgaa 60
agaacccacc tcaacatcaa accttttctt ggttttgtcc ctcatggaac cacatgtcta 120
tccgtacaat cttcttcgtg gaggaacgat ggaatggttg ctggagtttc atatccattt 180
tgtgcaaatt tctccagaag aagacagaga aaactttcaa ctcctaggag tcaaggctct 240
tcaccaaagg ggtttgtgcc aaggacgcct tcagggatga gcacacaaag aagggatcag 300
aagagcaacg gtgataaaga aagtcaaagt acttcatcat ccaaagaatc agaaatttcc 360
aaccccaaga cgtttgaagc aaaagttgaa actagtgatg atggcactaa acaagtggga 420
agggaccgca agtctcagga ggaggaggat gaattcaatg gtgccactaa atcagtaagt 480
ctatcacctg ttcgtggatc aactcaattt gttggaagtg gagaaactgg tgacaatgac 540
gtgggtgctg taaattttaa taaatcaaat agaacggaag agagtgattt tcaaattgat 600
tctgtaataa gagaacaaag tggaggtgat tactccgaaa atattcaggg gaaaactagc 660
aagggcagcc tcgctttggg tacaccactt tctgaaatat tgcagctaga tattgatgga 720
ggttataaag ttgaacaata caatcatgat gagttggatg agacacaaaa actgaaagaa 780
aatgatgctg ggaatgttga agacaaaaga cccttagcaa gagagctgtt ggaaatgact 840
aagcctagca atgtggaatt tactgaaagc aatgagatca ctgaagtaga cagtaatagt 900
ttcttaaaac cagattcagt tgacgagagt gagccatcaa ctgtaggaac attagagact 960
gaagatattt ctctgaagtt aagattggag atggaagcga atctacgtag gcaggctata 1020
aaaaggcttg ccgaggaaaa tttattgcaa ggaatcagat tattttgttt cccagaggtt 1080
gtaaaaccca atgaagatgt cgaaatattc cttaacagga gtctttccac tttgaataat 1140
gaacctgatc tcttgattat gggagctttt aatgactggc ggtggagatc ttttactaca 1200
acactaactg agactcatct cagtggagat tggtggtctt gcaagatcca cgttccgaag 1260
gaagcataca agattgattt tgtgtttttt aatggaaagg atgtctatga caacaatgat 1320
aataatgatt tcagtataac tgtggaaggt ggtatgcaaa ttcttgattt tgaaaatttc 1380
ttgctcgagg agaaacggag agaacaggag aaacttgcta aagaacaagc tgaaagagaa 1440
agactagcag aagaacaaag gcgaatagaa gcagagaaag ctgcacttga agctgataga 1500
tcacaggcaa aggaagaggc tgcaaagaaa agggaagtat tgcaagcact gatggcaaaa 1560
gcctcgaaga ctcgtgatat cacctggtat atcgaaccaa gtgtatttaa atgtgaggag 1620
aaggtcaagt tatactatga caaaagttca ggtcctctct cccatgctaa ggacttgtgg 1680
atccatggag gatataacaa ctggaaggat ggtttgtcta tcgttgaaaa gcttgttaaa 1740
tctgagagaa tagatggtga ttggtggtat acagaggttg tcatccctga tcgggcactt 1800
gtcttggatt gggtttttgc tgatggtcca cccaagcatg ccattgcata tgataacaat 1860
catcgccaag acttccatgc cattgtcccc aagcacattc cggaggaatt atattgggtt 1920
gaggaagaac ttcagatctt caaggcactt caggaggaga gaaggcttag agaagaggct 1980
atgcgtgcta aggctgaaaa agcagcacgt atgaaagctg aaacaaagga aaggactatg 2040
aaatcatttt tattgtctca gaagcacata gtgtacactg agcctcttga tgtccaagct 2100
ggaagcagcg tcacagttta ctataatccc gccaatacag tacttaatgg taaacctgaa 2160
atttggttca gatgttcatt caatcgttgg actcaccgcc tgggtccatt gccgcctcaa 2220
aaaatgttgc ctactgaaaa tggaacccat gtcaaagcaa ctgtgaaggt tccattggat 2280
gcacatatga tggattttgt attctctgag agagaagatg gtggaatttt tgacaataga 2340
agtggaatgg actatcacat acctgtgttt ggaggagtcg caaaagaacc tccgatgcat 2400
attgttcata ttgctgtcga aatggcacct attgcaaagg tgggaggcct tggtgatgtt 2460
gttactagtc tttcccgtgc agttcaagat ttaaaccata atgtggatat catcttacct 2520
aagtatgact gtttgaagat gaatcaggtg aaggactttc agtttcacaa aagctacttt 2580
tggggtggga ctgaaataaa agtatggttt gggaaggtgg aaggtgtctc cgtctacttt 2640
ttggagcctc aaaacgggtt attttggaag gggtgtgtat atggttgtaa taatgatggt 2700
gaaagatttg gtttcttttg tcacgcggct ttggagtttc ttctgcaagg tggatttcat 2760
ccggatatca tccattgcca tgattggtct agtgctcccg ttgcgtggct ctttaaggaa 2820
caatatacac actatggtct aagcaagtct cgaattgtct tcacaataca caatcttgaa 2880
tttggtgcag atctcattgg gaaagcaatg acttatgcag acaaagctac aacagtttcc 2940
ccaacttact caagggaggt gtctggaaac cctgtaattg cacctcacct ttacaagttc 3000
catggtatag tgaacgggat tgacccagat atttgggacc cgttgaacga taagttcatt 3060
ccgatttcat acacctcgga aaatgttgtt gaaggcaaaa cagcagccaa ggaagctttg 3120
cagcaaaaac ttggactgaa acaggccgac cttcctttgg taggaataat cacccgctta 3180
actcaccaga aaggaatcca cctcatcaaa catgctattt ggcgcaccct ggaacggaat 3240
ggacaggtgg tcttgcttgg ttctgctcct gatcctagga tacaaaatga ttttgttaat 3300
ttggcaaatc aattgcactc cacatataat gaccgtgcac ggctctgtct aacatacgat 3360
gagccacttt ctcacctgat atatgctggt gctgatttca ttctagtccc ttcaatattt 3420
gagccatgtg gactaacaca acttacggcg atgagatatg gttcaattcc aattgtgcgt 3480
aaaactggag gactttatga tactgtattt gatgttgacc atgacaaaga gagagcacaa 3540
cagtgtggtc ttgaaccaaa tggattcagc tttgatggag ctgatgctgc cggagttgat 3600
tatgctctga ataggcaatt atcttgttcc tcctaa 3636
<210> 2
<211> 1211
<212> PRT
<213> 人工序列(NtGLGA)
<400> 2
Met Asp Val Pro Leu Pro Leu Gln Arg Pro Leu Ser Tyr Ser Asn Ser
1 5 10 15
Val Ser Thr Glu Arg Thr His Leu Asn Ile Lys Pro Phe Leu Gly Phe
20 25 30
Val Pro His Gly Thr Thr Cys Leu Ser Val Gln Ser Ser Ser Trp Arg
35 40 45
Asn Asp Gly Met Val Ala Gly Val Ser Tyr Pro Phe Cys Ala Asn Phe
50 55 60
Ser Arg Arg Arg Gln Arg Lys Leu Ser Thr Pro Arg Ser Gln Gly Ser
65 70 75 80
Ser Pro Lys Gly Phe Val Pro Arg Thr Pro Ser Gly Met Ser Thr Gln
85 90 95
Arg Arg Asp Gln Lys Ser Asn Gly Asp Lys Glu Ser Gln Ser Thr Ser
100 105 110
Ser Ser Lys Glu Ser Glu Ile Ser Asn Pro Lys Thr Phe Glu Ala Lys
115 120 125
Val Glu Thr Ser Asp Asp Gly Thr Lys Gln Val Gly Arg Asp Arg Lys
130 135 140
Ser Gln Glu Glu Glu Asp Glu Phe Asn Gly Ala Thr Lys Ser Val Ser
145 150 155 160
Leu Ser Pro Val Arg Gly Ser Thr Gln Phe Val Gly Ser Gly Glu Thr
165 170 175
Gly Asp Asn Asp Val Gly Ala Val Asn Phe Asn Lys Ser Asn Arg Thr
180 185 190
Glu Glu Ser Asp Phe Gln Ile Asp Ser Val Ile Arg Glu Gln Ser Gly
195 200 205
Gly Asp Tyr Ser Glu Asn Ile Gln Gly Lys Thr Ser Lys Gly Ser Leu
210 215 220
Ala Leu Gly Thr Pro Leu Ser Glu Ile Leu Gln Leu Asp Ile Asp Gly
225 230 235 240
Gly Tyr Lys Val Glu Gln Tyr Asn His Asp Glu Leu Asp Glu Thr Gln
245 250 255
Lys Leu Lys Glu Asn Asp Ala Gly Asn Val Glu Asp Lys Arg Pro Leu
260 265 270
Ala Arg Glu Leu Leu Glu Met Thr Lys Pro Ser Asn Val Glu Phe Thr
275 280 285
Glu Ser Asn Glu Ile Thr Glu Val Asp Ser Asn Ser Phe Leu Lys Pro
290 295 300
Asp Ser Val Asp Glu Ser Glu Pro Ser Thr Val Gly Thr Leu Glu Thr
305 310 315 320
Glu Asp Ile Ser Leu Lys Leu Arg Leu Glu Met Glu Ala Asn Leu Arg
325 330 335
Arg Gln Ala Ile Lys Arg Leu Ala Glu Glu Asn Leu Leu Gln Gly Ile
340 345 350
Arg Leu Phe Cys Phe Pro Glu Val Val Lys Pro Asn Glu Asp Val Glu
355 360 365
Ile Phe Leu Asn Arg Ser Leu Ser Thr Leu Asn Asn Glu Pro Asp Leu
370 375 380
Leu Ile Met Gly Ala Phe Asn Asp Trp Arg Trp Arg Ser Phe Thr Thr
385 390 395 400
Thr Leu Thr Glu Thr His Leu Ser Gly Asp Trp Trp Ser Cys Lys Ile
405 410 415
His Val Pro Lys Glu Ala Tyr Lys Ile Asp Phe Val Phe Phe Asn Gly
420 425 430
Lys Asp Val Tyr Asp Asn Asn Asp Asn Asn Asp Phe Ser Ile Thr Val
435 440 445
Glu Gly Gly Met Gln Ile Leu Asp Phe Glu Asn Phe Leu Leu Glu Glu
450 455 460
Lys Arg Arg Glu Gln Glu Lys Leu Ala Lys Glu Gln Ala Glu Arg Glu
465 470 475 480
Arg Leu Ala Glu Glu Gln Arg Arg Ile Glu Ala Glu Lys Ala Ala Leu
485 490 495
Glu Ala Asp Arg Ser Gln Ala Lys Glu Glu Ala Ala Lys Lys Arg Glu
500 505 510
Val Leu Gln Ala Leu Met Ala Lys Ala Ser Lys Thr Arg Asp Ile Thr
515 520 525
Trp Tyr Ile Glu Pro Ser Val Phe Lys Cys Glu Glu Lys Val Lys Leu
530 535 540
Tyr Tyr Asp Lys Ser Ser Gly Pro Leu Ser His Ala Lys Asp Leu Trp
545 550 555 560
Ile His Gly Gly Tyr Asn Asn Trp Lys Asp Gly Leu Ser Ile Val Glu
565 570 575
Lys Leu Val Lys Ser Glu Arg Ile Asp Gly Asp Trp Trp Tyr Thr Glu
580 585 590
Val Val Ile Pro Asp Arg Ala Leu Val Leu Asp Trp Val Phe Ala Asp
595 600 605
Gly Pro Pro Lys His Ala Ile Ala Tyr Asp Asn Asn His Arg Gln Asp
610 615 620
Phe His Ala Ile Val Pro Lys His Ile Pro Glu Glu Leu Tyr Trp Val
625 630 635 640
Glu Glu Glu Leu Gln Ile Phe Lys Ala Leu Gln Glu Glu Arg Arg Leu
645 650 655
Arg Glu Glu Ala Met Arg Ala Lys Ala Glu Lys Ala Ala Arg Met Lys
660 665 670
Ala Glu Thr Lys Glu Arg Thr Met Lys Ser Phe Leu Leu Ser Gln Lys
675 680 685
His Ile Val Tyr Thr Glu Pro Leu Asp Val Gln Ala Gly Ser Ser Val
690 695 700
Thr Val Tyr Tyr Asn Pro Ala Asn Thr Val Leu Asn Gly Lys Pro Glu
705 710 715 720
Ile Trp Phe Arg Cys Ser Phe Asn Arg Trp Thr His Arg Leu Gly Pro
725 730 735
Leu Pro Pro Gln Lys Met Leu Pro Thr Glu Asn Gly Thr His Val Lys
740 745 750
Ala Thr Val Lys Val Pro Leu Asp Ala His Met Met Asp Phe Val Phe
755 760 765
Ser Glu Arg Glu Asp Gly Gly Ile Phe Asp Asn Arg Ser Gly Met Asp
770 775 780
Tyr His Ile Pro Val Phe Gly Gly Val Ala Lys Glu Pro Pro Met His
785 790 795 800
Ile Val His Ile Ala Val Glu Met Ala Pro Ile Ala Lys Val Gly Gly
805 810 815
Leu Gly Asp Val Val Thr Ser Leu Ser Arg Ala Val Gln Asp Leu Asn
820 825 830
His Asn Val Asp Ile Ile Leu Pro Lys Tyr Asp Cys Leu Lys Met Asn
835 840 845
Gln Val Lys Asp Phe Gln Phe His Lys Ser Tyr Phe Trp Gly Gly Thr
850 855 860
Glu Ile Lys Val Trp Phe Gly Lys Val Glu Gly Val Ser Val Tyr Phe
865 870 875 880
Leu Glu Pro Gln Asn Gly Leu Phe Trp Lys Gly Cys Val Tyr Gly Cys
885 890 895
Asn Asn Asp Gly Glu Arg Phe Gly Phe Phe Cys His Ala Ala Leu Glu
900 905 910
Phe Leu Leu Gln Gly Gly Phe His Pro Asp Ile Ile His Cys His Asp
915 920 925
Trp Ser Ser Ala Pro Val Ala Trp Leu Phe Lys Glu Gln Tyr Thr His
930 935 940
Tyr Gly Leu Ser Lys Ser Arg Ile Val Phe Thr Ile His Asn Leu Glu
945 950 955 960
Phe Gly Ala Asp Leu Ile Gly Lys Ala Met Thr Tyr Ala Asp Lys Ala
965 970 975
Thr Thr Val Ser Pro Thr Tyr Ser Arg Glu Val Ser Gly Asn Pro Val
980 985 990
Ile Ala Pro His Leu Tyr Lys Phe His Gly Ile Val Asn Gly Ile Asp
995 1000 1005
Pro Asp Ile Trp Asp Pro Leu Asn Asp Lys Phe Ile Pro Ile Ser Tyr
1010 1015 1020
Thr Ser Glu Asn Val Val Glu Gly Lys Thr Ala Ala Lys Glu Ala Leu
1025 1030 1035 1040
Gln Gln Lys Leu Gly Leu Lys Gln Ala Asp Leu Pro Leu Val Gly Ile
1045 1050 1055
Ile Thr Arg Leu Thr His Gln Lys Gly Ile His Leu Ile Lys His Ala
1060 1065 1070
Ile Trp Arg Thr Leu Glu Arg Asn Gly Gln Val Val Leu Leu Gly Ser
1075 1080 1085
Ala Pro Asp Pro Arg Ile Gln Asn Asp Phe Val Asn Leu Ala Asn Gln
1090 1095 1100
Leu His Ser Thr Tyr Asn Asp Arg Ala Arg Leu Cys Leu Thr Tyr Asp
1105 1110 1115 1120
Glu Pro Leu Ser His Leu Ile Tyr Ala Gly Ala Asp Phe Ile Leu Val
1125 1130 1135
Pro Ser Ile Phe Glu Pro Cys Gly Leu Thr Gln Leu Thr Ala Met Arg
1140 1145 1150
Tyr Gly Ser Ile Pro Ile Val Arg Lys Thr Gly Gly Leu Tyr Asp Thr
1155 1160 1165
Val Phe Asp Val Asp His Asp Lys Glu Arg Ala Gln Gln Cys Gly Leu
1170 1175 1180
Glu Pro Asn Gly Phe Ser Phe Asp Gly Ala Asp Ala Ala Gly Val Asp
1185 1190 1195 1200
Tyr Ala Leu Asn Arg Gln Leu Ser Cys Ser Ser
1205 1210
Claims (7)
1.一种烟草淀粉合酶基因,其特征在于,碱基序列具体如SEQ ID NO.1所示。
2.根据权利要求1所述的烟草淀粉合酶基因,其特征在于,烟草淀粉合酶基因的氨基酸序列如SEQ ID NO.2所示。
3.根据权利要求1或2所述的烟草淀粉合酶基因,其特征在于,烟草淀粉合酶基因的PCR扩增制备方法,包括如下步骤:
(1)提取基因组,并反转录为cDNA备用;
(2)设计PCR扩增用引物,并进行PCR扩增,具体引物序列设计如下:
NtGLGA-F:5’-TTGGAGTTTCTTCTGCAAGGTG-3’,
NtGLGA-R:5’-CAATTACAGGGTTTCCAGACAC-3’。
4.根据权利要求3所述的烟草淀粉合酶基因,其特征在于,在步骤(1)中提取基因组时,以烟草品种红花大金元叶片为样品。
5.一种烟草淀粉合酶基因的应用,其特征在于,利用上述权利要求1至4中任一项的烟草淀粉合酶基因,该基因表达的蛋白与烟草叶片中淀粉含量相关,降低该蛋白表达后,叶片中支链淀粉和总淀粉含量明显降低。
6.根据权利要求5所述的烟草淀粉合酶基因的应用,其特征在于,利用基因沉默技术、或者基因超表达方法,通过调节烟草淀粉合酶基因的表达量,来调节控制烟叶中淀粉含量。
7.根据权利要求6所述的烟草淀粉合酶基因的应用,其特征在于,通过转基因技术、瞬时表达技术或基因组编辑技术,构建含有烟草淀粉合酶基因的病毒诱导沉默载体、RNAi干涉载体、超表达载体或基因组编辑载体,转化烟草,筛选获得淀粉含量变化的烟草新品种。
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Citations (3)
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US5981728A (en) * | 1997-11-12 | 1999-11-09 | Iowa State University Research Foundation | Dull1 coding for a novel starch synthase and uses thereof |
CN107759676A (zh) * | 2017-11-27 | 2018-03-06 | 南京农业大学 | 一种植物直链淀粉合成相关蛋白Du15与其编码基因及应用 |
CN110938639A (zh) * | 2019-12-19 | 2020-03-31 | 中国烟草总公司郑州烟草研究院 | 烟草ATP合酶γ链NtATPG及其应用 |
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US5981728A (en) * | 1997-11-12 | 1999-11-09 | Iowa State University Research Foundation | Dull1 coding for a novel starch synthase and uses thereof |
CN107759676A (zh) * | 2017-11-27 | 2018-03-06 | 南京农业大学 | 一种植物直链淀粉合成相关蛋白Du15与其编码基因及应用 |
CN110938639A (zh) * | 2019-12-19 | 2020-03-31 | 中国烟草总公司郑州烟草研究院 | 烟草ATP合酶γ链NtATPG及其应用 |
Non-Patent Citations (1)
Title |
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PREDICTED: Nicotiana tabacum soluble starch synthase 3, chloroplastic/amyloplastic-like (LOC107770809), transcript variant X1, mRNA;Zhang, H.等;GENBANK;1-2 * |
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