CN117604003A - 一个大豆天冬氨酸激酶/高丝氨酸脱氢酶家族基因GmAK-HSDH的应用 - Google Patents
一个大豆天冬氨酸激酶/高丝氨酸脱氢酶家族基因GmAK-HSDH的应用 Download PDFInfo
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Abstract
本发明公开了一个大豆天冬氨酸激酶/高丝氨酸脱氢酶家族基因GmAK‑HSDH的应用。大豆GmAK‑HSDH蛋白编码基因GmAK‑HSDH，其核苷酸序列为：SEQ ID NO.1。将构建的过表达载体pBA002‑GmAK‑HSDH利用子叶节转化法转入受体材料JACK。表明该基因可以作为目的基因导入大豆植株，通过GmAK‑HSDH基因的过量表达，使转基因大豆种子水溶蛋白和蛋白质含量显著提高。可见,本发明所述的大豆GmAK‑HSDH蛋白编码基因GmAK‑HSDH可通过基因工程手段在提高大豆种子蛋白加工品质，培育高蛋白大豆品种方面的应用。
Description
技术领域
本发明属于植物基因工程领域,涉及一个大豆天冬氨酸激酶/高丝氨酸脱氢酶家族基因GmAK-HSDH的应用，具体涉及来源于栽培大豆，编码天冬氨酸激酶(AK)和高丝氨酸脱氢酶(HSDH)两大功能结构域基因GmAK-HSDH在调控大豆籽粒水溶蛋白和蛋白质含量相关方面的应用。
背景技术
栽培大豆(Glycine max(L.)Merr.)是一种广泛分布于世界各地的重要的种子作物，最初是从野生大豆(G.soja Sieb.and Zucc.)驯化而来。大豆作为一种主要的油料种子作物，为食品、饲料和工业应用提供了有价值的植物油和蛋白质。大豆蛋白通常易溶于水，而传统大豆食品中只能加工和利用水溶性蛋白，因此水溶蛋白含量成为大豆食品加工的重要指标。
天冬氨酸衍生氨基酸的生物合成第一步是通过单功能天冬氨酸激酶(AKs)和双功能天冬氨酸激酶/高丝氨酸脱氢酶(AK-HSDHs)将天冬氨酸转化为天冬氨酸-4-磷酸。接着天冬氨酸-4-磷酸转化为天冬氨酸半醛，分支点的中间产物用于Lys或Met，Thr和Ile的生物合成。其中一条大分支为Met,Thr和Ile的生物合成，关键步骤是由双功能AK-HSDHs催化天冬氨酸半醛形成同源丝氨酸。高等植物有多种编码AK和AK-HSDH的基因。例如，拟南芥(Arabidopsis thaliana)核基因组包含3个AK编码基因(AK1[At5g13280]、AK2[A5g14060]和AK3[At3g02020])和2个AK-HSDH编码基因(AK-HSDH1[At1g31230]和AK-HSDH2[At4g19710])(Jander and Joshi2009)。公开的微阵列数据表明，这五种酶在所有植物组织中都有表达。这些酶的转录是在黑暗和其他低糖条件下被基本亮氨酸拉链转录因子抑制。此外，AKs和AK-HSDHs都受到下游产品的反馈抑制。例如，单功能AKs的活性被Lys反馈抑制是通过蛋白C端两个赖氨酸结合的ACT结构域介导的。AK-HSDHs的活性被Thr反馈抑制是通过蛋白中间的两个Thr结合的ACT结构域介导的。Teresa和Lu通过对一系列AK和AK-HSDH单、双功能缺失突变体的游离和蛋白结合氨基酸谱分析、转录丰度和活性分析。最终发现，AK总活性与HSDH总活性之比与Lys与Met、Thr、Ile总量之比呈负相关。Lys敏感的单功能AKs和Thr敏感的双功能AK-HSDHs之间的平衡对维持天冬氨酸及其衍生氨基酸的水平和比例具有重要意义。
发明内容
本发明的目的在于提供公开编码天冬氨酸激酶和高丝氨酸脱氢酶两大功能结构域基因GmAK-HSDH的高品质基因工程应用。GmAK-HSDH基因在大豆各组织中均有表达，亚细胞定位显示GmAK-HSDH编码的蛋白定位在叶绿体。在大豆中过表达GmAK-HSDH基因提高了大豆种子的水溶蛋白和蛋白质含量。
GmAK-HSDH作为目的基因通过基因工程手段在受体材料中过量表达该基因从而改变大豆种子水溶蛋白和蛋白质含量。
本研究通过测定由南京农业大学国家大豆改良中心提供，来自于中国三大大豆主产区的25个省份，以及海外大豆主产区：美国，巴西等国家的272份栽培品种组成的自然群体在三个环境中水溶蛋白含量，结合重测序标记进行全基因组关联分析，在8号染色体显著关联的SNP位点发掘出一个编码天冬氨酸激酶和高丝氨酸脱氢酶两大功能结构域基因Glyma.08G107800，可能影响大豆籽粒水溶蛋白含量。AK-HSDH是一个双功能的天冬氨酸激酶/高丝氨酸脱氢酶，在天冬氨酸家族氨基酸合成通路中是重要的限速酶之一。
本发明的目的可通过如下技术方案实现:
大豆双功能酶编码基因GmAK-HSDH在基因工程改造大豆种子中水溶蛋白和蛋白质含量的应用，所述的大豆双功能酶编码基因GmAK-HSDH，编码区序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO.2所示。
在大豆中，过表达GmAK-HSDH基因提高了种子的水溶蛋白和蛋白质含量。
有益效果:
本发明中GmAK-HSDH编码双功能的天冬氨酸激酶/高丝氨酸脱氢酶。通过组织表达分析发现GmAK-HSDH在大豆所有组织中均表达，在叶片和35d种子中的表达量较高，亚细胞定位显示GmAK-HSDH蛋白于叶绿体中被定位到。大豆品种JACK中过表达GmAK-HSDH，发现与对照相比，转基因大豆种子的水溶蛋白和蛋白质含量有所提高。转基因大豆种子的水溶蛋白和蛋白质含量统计分析表明GmAK-HSDH过表达转基因株系与对照相比能够显著提高大豆种子的水溶蛋白和蛋白质含量。本发明公开了该基因在调控大豆品质性状中的效用，可以通过定向地改造作物的水溶蛋白和蛋白质含量，提高作物的品质。
利用植物过表达载体pBA002-GmAK-HSDH,将本发明的GmAK-HSDH导入植物体内,可以调控植株种子的品质性状，获得转基因植株。
附图说明
下面结合附图及实施例对本发明做进一步说明。
图1GmAK-HSDH基因的克隆。根据Phytozome网站预测的GmAK-HSDH序列信息设计引物，以栽培大豆材料NJAU_089的叶片cDNA为模板PCR扩增，得到一条长2751bp的DNA片段。经过测序结果分析，该片段的序列信息与Phytozome网站预测的序列一致，即该2751bp片段即为GmAK-HSDH基因。Marker：5k，从下往上依次为300，500，800，1000，1500，2000，3000，5000bp。
图2GmAK-HSDH基因的组织表达分析。采用实时荧光定量PCR技术对GmAK-HSDH在栽培大豆材料NJAU_089的不同组织表达进行研究，栽培大豆不同组织分别为根、茎、叶、花、7d荚、14d、21d、28d和35d种子。
图3GmAK-HSDH的亚细胞定位。GFP代表绿色荧光蛋白；Chl代表了叶绿体自发荧光；BF代表的是明场；Merge则代表各通道的信号叠加合成；标尺为50μm。
图4T0代过表达转基因植株的bar试纸条检测。1代表JACK，2-4分别代表3个T0代阳性单株。图5T2代过表达转基因植株的阳性鉴定及GmAK-HSDH的相对表达水平。A图：T2代转基因大豆株系中目的基因的PCR检测。使用特异性检测引物对扩增部分载体序列和GmAK-HSDHCDS序列。Marker：2k，从下往上依次为100，250，500，750，1000，2000bp。P：阳性质粒。N：阴性对照。B图：GmAK-HSDH基因在不同株系中的表达水平检测。相对表达水平是经tubulin基因标准化后相对对照的表达量(CK中的相对表达水平＝1)。OE-3,OE-4和OE-6为不同的转基因株系，JACK为对照的受体材料。*表示0.01<p<0.05水平下显著差异；**表示p<0.01水平下极显著差异。
图6转基因大豆种子水溶蛋白含量统计分析。OE-3,OE-4和OE-6为不同的转基因株系，JACK为对照的受体材料。*表示0.01<p<0.05水平下显著差异；**表示p<0.01水平下极显著差异。
图7转基因大豆种子蛋白质含量统计分析。OE-3,OE-4和OE-6为不同的转基因株系，JACK为对照的受体材料。*表示0.01<p<0.05水平下显著差异；**表示p<0.01水平下极显著差异。
具体实施方式
下面结合附图和实施例,并参照数据进一步详细地描述本发明。这些实施例只是为了举例说明本发明,而非以任何方式限制本发明的范围。在以下的实施例中,未详细描述的各种过程和方法是本领域中公知的常规方法。所用到的引物,均在首次出现时标明,其后所用相同引物,均以首次标明的内容相同。
实施例1大豆双功能酶基因GmAK-HSDH的克隆与鉴定
以南京农业大学国家大豆改良中心提供的NJAU_089实验材料，2021年6月种植于南京农业大学牌楼教学实验基地，以苗期的叶片cDNA为模板，以GmAK-HSDH-F和GmAK-HSDH-R为引物进行PCR扩增。
上游引物GmAK-HSDH-F：TCGTCGTTTCACTTCGTTTC；(SEQ ID NO.3)
下游引物GmAK-HSDH-R：TTCAGATAATGCGTGTCGTG。(SEQ ID NO.4)
应用RT-PCR方法，从大豆叶器官总RNA中扩增GmAK-HSDH基因。取大豆叶组织，用研钵研碎，加入盛有裂解液的1.5mL EP管，充分振荡后，再移入玻璃匀浆器内。匀浆后移至1.5mL EP管中，采用新型植物总RNA提取试剂盒(天根)的说明书步骤提取大豆组织的RNA。用甲醛变性胶电泳鉴定总RNA质量，然后在分光光度计上测定RNA含量。以获得的总RNA为模板,按照IIQ RT SuperMix for qPCR(+gDNA wiper)试剂盒(Vazyme)说明书进行反转录，合成cDNA第一链。进行PCR扩增反应。PCR反应体系为:2μl cDNA(0.05μg)、上、下游引物各2μl(10μM)、25μl 2×Phanta Max Buffer、1μl dNTP(10mM)和1U PhantaMax Super-Fidelity DNA聚合酶(Vazyme),用超纯水补足50μl。PCR程序如下：在Bio-RAD PTC200型PCR仪上进行,其程序为95℃预变性3min；95℃变性15s，58℃退火15s，72℃延伸95s,共35个循环；然后72℃延伸5min终止反应，4℃保存。PCR产物回收将其克隆至pCE2 TA/Blunt-Zero载体，测序后获得具有完整编码区的大豆基因GmAK-HSDH的cDNA序列SEQ ID NO.1，全长2751bp，编码SEQ ID NO.2所示的916个氨基酸。
实施例2GmAK-HSDH在栽培大豆不同器官中的表达特征
提取NJAU_089材料的根、茎、叶、花、7d荚、14d、21d、28d和35d种子的RNA，反转成cDNA进行RT-PCR分析。
总RNA的提取同实施例1。以大豆组成型表达基因Tubulin为内参基因，其扩增引物为Tubulin正向引物序列:CCTCGTTCGAATTCGCTTTTTG，Tubulin反向引物序列:CAACTGTCTTGTCACTTGGCAT。以来自大豆不同组织或器官的cDNA为模板，进行实时荧光定量PCR分析。GmAK-HSDH的扩增引物为GmAK-HSDH-qPCR-F:ACCTTCTCACACTTCGCTCC,GmAK-HSDH-qPCR-R:GGGTAGTTGTTTTTCCTCCA。结果(图2)分析表明GmAK-HSDH在大豆全生育期的各个组织中均有表达，其中在叶片和35d种子中表达量较高，并且在种子发育期间表现出表达量升高的趋势，这可能表明GmAK-HSDH对大豆籽粒的发育有一定的影响。
实施例3GmAK-HSDH的亚细胞定位
亚细胞定位采用本氏烟草瞬时表达的方法，使用的载体是P2，引物为GmAK-HSDH-P2-F:ACAAATCTATCTCTCTCGAGATGGCGTCGTTTTCCGCCGC，GmAK-HSDH-P2-R:GCTCACCATGGATCCCGATGGAGCACCAAGATATG。PCR扩增，目的条带正确后进行割胶回收，胶回收产物通过同源重组的方法连接到载体上，构建亚细胞定位载体P2-GmAK-HSDH(基因在GFP的N端)，烟草瞬时表达表达后暗培养48h，经激光共聚焦显微镜(Zeiss，LSM780)激光照射后，可产生绿色荧光信号，对蛋白质进行定位，观察拍照。结果如图3-5所示，转入空载质粒在整个细胞中都有分布，GmAK-HSDH：GFP融合蛋白集中分布于叶绿体上，表明Gm AK-HSDH可能主要通过富含叶绿体的器官，如叶片来发挥功能。
实施例4基因GmAK-HSDH的基因工程应用
1)过表达载体pBA002-GmAK-HSDH的构建
创制过表达转基因大豆使用的载体是pBA002，引物为GmAK-HSDH-pBA002-F:CGCGCCGGGCCCAGGCCTACGCGTATGGCGTCGTTTTCCGCCGC(SEQ ID NO.5)，GmAK-HSDH-pBA002-R:ATCGGGGAAATTCGAGCTCTTACGATGGAGCACCAAGAT(SEQ ID NO.6)。PCR扩增，目的条带正确后进行切胶回收，胶回收产物通过同源重组的方法连接到载体上，其中将pBA002载体利用Mlul和SacⅠ限制性内切酶进行双酶切，然后重组连接，产物即表达载体pBA002-GmAK-HSDH，转化大肠杆菌DH5α,转化液涂布于含50mg/LKana的LB固体培养基上筛选阳性克隆。经测序验证后,提取质粒,得到pBA002-GmAK-HSDH植物过量表达载体,用冻融法将pBA002-GmAK-HSDH转入根癌农杆菌菌株EHA105中。
2)植物过表达载体及敲除载体遗传转化大豆
利用由农杆菌株EHA105介导的子叶节转化法创制转基因大豆植株，具体的创制过程如下：
挑选出表面无任何缺陷、颗粒饱满、大小种皮颜色均匀一致的大豆种子，在通风橱中进行灭菌。其中发生化学反应HCl(浓)+NaClO→Cl2↑+NaOH(浓盐酸与次氯酸钠体积约1:10的比例)产生的氯气来进行消毒。实验中，取120ml的NaClO放入锥形瓶中。此时将放在培养皿的豆子放入烘干器中，将锥形瓶放入烘干器的正中间，盖上盖子。再将15ml浓HCl通过分液漏斗从烘干器上方缓慢加入，消毒时间6-7小时。
种子萌发：灭菌后的种子在超净工作台中充分吹散氯气，垂直插入预先配好凝固的SG4萌发固体培养基中，使培养基没过种脐一半即可。
接菌：在锥形瓶中加120ml左右加抗生素Kan和Rif的YEB液体培养基，加入1-2ml小量菌液，于28℃，200rpm摇至OD600＝0.85-0.9。
农杆菌侵染：将摇好的菌液室温下5000rpm离心10min，弃上清，然后两个离心管内加入共培养液CCM振荡悬浮，并调整至在OD600＝0.5-0.6。大豆种子萌发5天后，切除部分下胚轴保留5-10mm，再沿子叶和下胚轴将种子切开，去除真叶，然后在子叶节部分用刀顺着下胚轴的方向轻轻划出几道伤口。将处理好的外植体和悬浮好的菌液一起倒入灭过菌的罐子中，于28℃，120rpm共培养30-40min。最后将外植体取出，子叶节一面向下，置于铺有一层滤纸的固体共培养基CCM上，每个培养皿内放14个外植体，25℃黑暗培养5天。
丛生芽的诱导：将共培养5天后的外植体用灭菌水和Wash-Liquid除菌后，切掉过长的下胚轴，留大约5-10mm，生长点向上45°斜插入到不含草丁膦的SIM固体培养基中，每皿8个，26℃光照培养14-17天。14-17天后切除大芽和部分下胚轴，将长出丛生芽的外植体更换到加入6mg/L草丁膦的SIM固体培养基中进行筛选，培养14-17天。
伸长：将未完全枯死的外植体的子叶、枯叶和部分下胚轴切除，更换到加入4mg/L草丁膦的SEM固体培养基中培养14-17天。以14-17天为一个周期，切除枯叶和部分下胚轴，更换到新的SEM固体培养基，并逐渐降低草丁膦的浓度。
生根：当外植体的芽伸长到大约6cm时，切掉底部，在茎底部切一个十字型伤口，转入生根培养基RM中培养，约10天后可见诱导出的根。
炼苗：向瓶中倒入适量的无菌水，26℃光照培养约5天。移栽。当根的数量和长度适中时，将组培苗从培养基中分离，移入灭菌后的土中，置于人工培养箱中生长(16h光照/8h黑暗，25℃)。
过表达载体pBA002-GmAK-HSDH载体序列中含有筛选标记基因bar，在完成T0代苗的移栽后使用bar快速检测试纸条对T0代大豆植株进行阳性检测(图4)。待T0代苗长出新叶后，取不同节位叶片进行混检，加入500ul的缓冲液后将叶片捣碎磨浆并且混匀，之后将试纸条垂直插入混合液当中，待混合液体上升至试纸条一定高度后进行结果读取。若只有上面一条带则为阴性，若显示为两条带则表示样品为阳性，此时bar蛋白可以被检测到，认为该样品植株为阳性植株。提取初步筛选得到具有bar抗性的T0代转基因植株的基因组DNA,使用载体和基因特异性引物35s-F:GCTCCTACAAATGCCATCATTGC,和GmAK-HSDH-qPCR-R:GGGTAGTTGTTTTTCCTCCA进行PCR鉴定。结果表明得到的3个转基因株系均为成功转化过表达载体的大豆阳性植株(图5A)。经过实验室环境下的生长加代，在T2代,以GmAK-HSDH-qPCR-F：ACCTTCTCACACTTCGCTCC和GmAK-HSDH-qPCR-R：GGGTAGTTGTTTTTCCTCCA为引物,进行实时荧光定量PCR检测。结果表明与受体JACK相比，转基因株系OE-3,OE-4和OE-6中GmAK-HSDH的表达量极显著提高(图5B)。
3)转基因大豆种子水溶蛋白和蛋白质含量测定
经组培实验后共获得3个阳性过表达株系，实验室环境下繁殖加代后，在网室种植T2代植株并收获T3代的种子，种子放置28℃烘箱，一周后使用近红外光谱仪测定种子的水溶蛋白和蛋白质含量。三个过表达株系的种子水溶蛋白含量与受体JACK相比均提高，其中OE-4和OE-6株系达到了极显著水平。对成熟后种子蛋白质含量测定的结果表明，三个过表达株系的种子蛋白质含量相较于JACK也表现出提高的趋势，其中OE-3和OE-4株系达到了极显著水平。综上所述，GmAK-HSDH过表达能够提高大豆种子中水溶蛋白和蛋白质的含量，为高产优质新品种的选育创造了种质资源，对高品质大豆的培育提供了新思路。
Claims (5)
1.天冬氨酸激酶/高丝氨酸脱氢酶家族基因GmAK-HSDH，其核苷酸序列为：SEQ IDNO.1。
2.权利要求1所述的基因编码的GmAK-HSDH蛋白，其氨基酸序列为：SEQ ID NO.2。
3.含有权利要求1所述的GmAK-HSDH蛋白编码基因的重组表达载体。
4.权利要求1所述的GmAK-HSDH蛋白编码基因在增加大豆种子水溶蛋白和蛋白质含量中的应用。
5.权利要求3所述的重组表达载体在增加大豆种子水溶蛋白和蛋白质含量中的应用。
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