CN114164187B - 一种茶树咖啡碱转运蛋白及其编码基因和应用 - Google Patents
一种茶树咖啡碱转运蛋白及其编码基因和应用 Download PDFInfo
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- CN114164187B CN114164187B CN202111353764.5A CN202111353764A CN114164187B CN 114164187 B CN114164187 B CN 114164187B CN 202111353764 A CN202111353764 A CN 202111353764A CN 114164187 B CN114164187 B CN 114164187B
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Abstract
一种茶树咖啡碱转运蛋白CsPUP10.1及其编码基因和应用，属于生物基因工程技术领域。本发明一方面提供了茶树咖啡碱转运蛋白CsPUP10.1及其编码基因，另一方面提供了茶树咖啡碱转运蛋白编码基因的用途。本发明新发现了茶树咖啡碱转运蛋白CsPUP10.1，将该蛋白对应的基因在酵母中过表达，可以减轻咖啡碱对细胞的毒害作用，增强酵母对咖啡碱的耐受能力。
Description
技术领域
本发明属于生物基因工程技术领域，具体涉及一种茶树咖啡碱转运蛋白CsPUP10.1及其编码基因和应用。
背景技术
咖啡碱(caffeine)，是植物中一类重要的嘌呤生物碱，也是茶树中特异的次生代谢产物之一，占总生物碱含量的95％以上。茶树中的咖啡碱主要分布于叶片，占总干重的2％-3％，是茶叶品质和风味形成的主要成分。由于咖啡碱对人体具有兴奋、利尿等功效，还被广泛应用于药物和食品的生产研发。
研究发现，嘌呤透性酶(purine permeases,PUPs)是一类对嘌呤具有高亲和性的能量依赖型质子耦合转运蛋白，参与了细胞分裂素、生物碱、维生素B6等多种嘌呤类衍生物在植物体内的转运过程。生物碱合成后的转运，一方面可以缓解其对细胞的毒害作用；另一方面还可反馈调节其合成途径，有利于生物碱在植物体内的积累。在烟草、罂粟等含有生物碱的植物中，PUPs转运生物碱的功能均已得到证实。
目前，有关茶树咖啡碱转运的基因及蛋白质未见任何报道。鉴于茶树中含有丰富的咖啡碱，其转运过程是咖啡碱累积的重要环节，因此CsPUP10.1基因的发现对于研究茶树咖啡碱代谢途径具有重要的意义，也为以后利用微生物发酵生产咖啡碱奠定基础。
发明内容
针对现有技术存在的问题，本发明的目的在于设计提供一种茶树咖啡碱转运蛋白CsPUP10.1及其编码基因和应用的技术方案。
所述的一种转运咖啡碱的蛋白质，其特征在于该蛋白质的氨基酸序列为：
1)SEQ ID No.2所示的氨基酸序列；或
2)SEQ ID No.2所示的氨基酸序列经替换、缺失和/或添加一个或几个氨基酸残基形成的具有同等功能的氨基酸序列。
所述的编码蛋白质的基因,其特征在于该基因的核苷酸序列为：
1)SEQ ID No.1所示的核苷酸；或
2)SEQ ID No.1所示的核苷酸序列经取代一个或几个核苷酸，得到编码CsPUP10.1的核苷酸序列。
所述编码基因的重组载体。
所述的编码基因在调控茶树叶片咖啡碱含量中的应用。
所述的编码基因在提高酵母咖啡碱耐受性中的应用。
所述的一种制备咖啡碱耐受性提高的转基因酵母的方法，其特征在于包括如下步骤：将编码基因导入咖啡碱转运功能缺陷型酵母突变体fcy2中，使该基因过量表达，以减轻咖啡碱对细胞的毒害作用，增强酵母对咖啡碱的耐受能力，所述的编码基因的核苷酸序列为：
1)SEQ ID No.1所示的核苷酸；或
2)SEQ ID No.1所示的核苷酸序列经取代一个或几个核苷酸，得到编码CsPUP10.1的核苷酸序列。
本发明的实验证明，本发明新发现了茶树咖啡碱转运蛋白CsPUP10.1，将该蛋白对应的基因在酵母中过表达，可以减轻咖啡碱对细胞的毒害作用，增强酵母对咖啡碱的耐受能力。
附图说明
图1为茶树CsPUP10.1的基因和氨基酸序列；
图2为茶树不同组织中CsPUP10.1基因qPCR相对定量结果；
图3为不同茶树品种叶片中咖啡碱含量；
图4为不同茶树品种叶片中CsPUP10.1基因qPCR相对定量结果；
图5为0.3％咖啡碱处理下转基因酵母(fcy2+CsPUP10.1)与空载酵母(fcy2+EV)的表型比较。
具体实施方式
以下结合实施例来进一步说明本发明。
实施例
(1)基因克隆：以龙井43的叶片为材料，用试剂盒法提取总RNA；根据CsPUP10.1的mRNA序列设计引物，用反转录PCR法获得该基因的全长，并测序验证。最终获得茶树CsPUP10.1的基因序列，如图1所示，其核苷酸序列如SEQ ID No.1所示，其蛋白质的氨基酸序列如SEQ ID No.2所示。
上述反转录基因克隆所采用的引物为CsPUP10.1-CDS-F:5'-CAGCCTAAACAAGTCAACAACC-3(如SEQ ID No.3所示)；CsPUP10.1-CDS-R:5'-CAAAACAACATCACCTCAAAC-3'(如SEQ ID No.4所示)。
反应体系为50μL，包含25μL rTaq Master Mix，1-5μL cDNA，上下游引物各2μL，ddH2O补足至50μL。
反应条件为94℃，10分钟；94℃，30秒；53℃，30秒；72℃，1分钟；35个循环；72℃，10分钟；4℃保存。
(2)实时荧光定量分析：荧光定量PCR采用Roche LightCycler 480实时荧光定量PCR系统，以SYBR Green染料进行标记。内参基因选择茶树GAPDH基因(GE651107)。采用的引物为GAPDH-F:5′-TTGGCATCGTTGAGGGTCT-3′(如SEQ ID No.5所示)；GAPDH-R:5′-CAGTGGGAACACGGAAAGC-3′(如SEQ ID No.6所示)；CsPUP10.1-F:5′-ACGCAGGCTCTGATAACACGACA-3′(如SEQ ID No.7所示)；CsPUP10.1-R:5′-TTACCCGTTGGAAAGAGAGTTGG-3′(如SEQ ID No.8所示)。
反应体系为10μl，包含5μL SYBR premix，1μL模板cDNA，上下游引物各0.2μL,ddH2O补足至10μL。
反应条件为94℃，30秒；95℃，5秒；60℃，30秒；72℃，5秒；45个循环；95℃，5秒；60℃，1分钟保存。每组样品3个重复。如图2所示，茶树幼嫩叶、成熟叶、茎、根等不同组织部位CsPUP10.1基因qPCR相对定量结果，表明茶树CsPUP10.1基因在成熟叶中表达量最高。
(3)CsPUP10.1基因表达量与咖啡碱含量的相关性分析：以六个茶树品种的幼嫩叶、成熟叶为材料，包括‘龙井43’(longjing 43，LJ43)，‘中茶108’(zhongcha 108,ZC108)，‘中茗7号’(zhongming 7,ZM7)，‘中白4号’(zhongbai 4,ZB4)，‘中黄1号’(zhonghuang 1,ZH1)，‘恩施黄化’(enshi 4,ESN4)。部分材料用于qPCR基因表达量的检测，方法同上。部分材料用于咖啡碱含量的测定，具体步骤包括：准确称取0.2g茶样于10mL离心管中，加70％甲醇溶液5mL，70℃水浴浸提10min，冷却至室温，转入离心机在3500r/min转速下离心10min，同法操作两次，合并提取液定容至10mL容量瓶，摇匀。吸取供试样品提取液2mL至15mL离心管中，加入8mL稳定液，过0.45μm有机微孔滤膜，过滤至液相专用进样瓶中备用。HPLC检测方法：色谱柱为Phenomenex C12色谱柱(4.6mm×250mm，5μm)；流动相：1％甲酸水溶液(A)-乙腈(B)，线性梯度洗脱：0～42min，4％～18.7％B，42～43min，18.7％～4％B；柱温40℃，流速1.0mL·min-1，检测波长280nm；进样量10μL。试验结果表明，六个不同茶树品种叶片中咖啡碱含量与CsPUP10.1基因表达水平呈极显著负相关。如图3所示，不同茶树品种叶片中咖啡碱含量。如图4所示，不同茶树品种叶片中CsPUP10.1基因qPCR相对定量结果。
(4)酵母表达载体的构建及转化：将茶树CsPUP10.1全长cDNA克隆到酵母表达载体pYES2，并转入咖啡碱转运功能缺陷型酵母突变体fcy2中，进行基因功能验证。具体步骤包括：设计特异引物：正向引物5’-CCGCTCGAG(XhoI)ATGGAGTCTGCTCAAGAACTGG(如SEQ IDNo.9所示)和反向引物5’-TGCTCTAGA(XbaI)TCAACATAGCTCTATCCGAGCT(如SEQ ID No.10所示)。通过PCR方法扩增出目的基因CsPUP10.1的编码区序列；CsPUP10.1的PCR产物经切胶回收纯化后，分别对目的片段和酵母表达载体pYES2进行XhoI和XbaI双酶切；然后用T4 DNA连接酶将双酶切回收产物连接至载体上，连接产物转化到大肠杆菌DH5α感受态细胞中，经菌液PCR及测序验证后，提取得到重组质粒；根据酵母转化试剂盒说明书进行酵母转化实验，经阳性转化子鉴定后的转基因酵母即可用于后续试验。
(5)CsPUP10.1转基因酵母咖啡碱耐受性：以重组酵母fcy2+EV及fcy2+CsPUP10.1为材料。挑取单菌落于SD/-Ura液体筛选培养基(coolaber,PM2271)中，30℃，200rpm培养OD600值至0.5-1；测定菌液OD600值，并统一调整为0.5，取相应体积的菌液12000rpm离心1min，弃上清；加入1mL 0.9％Nacl重悬菌液，按照10倍梯度依次稀释至10-1、10-2、10-3、10-4，分别取菌液5μL进行点样，或分别取10-2稀释浓度的菌液50μL分半涂布于含0.3％咖啡碱处理的诱导培养基(每100ml含0.67g YNB，0.077g DO supplement-Ura，2g琼脂粉，2g半乳糖，1g棉子糖和0.3g咖啡碱)中；以fcy2+EV作为对照，30℃培养，观察酵母生长表型差异。试验结果如图5所示，当未添加咖啡碱时，转基因fcy2+CsPUP10.1与空载酵母fcy2+EV的生长无明显差异；而在0.3％咖啡碱处理下，与空载酵母fcy2+EV相比，转基因酵母fcy2+CsPUP10.1表现出明显的生长优势，对咖啡碱的耐受能力提高。
序列表
<110> 中国农业科学院茶叶研究所
<120> 一种茶树咖啡碱转运蛋白及其编码基因和应用
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tggcttcgag tgtcgctcta catcttcttc ctccttgccg gccaatcagc agccgtactt 180
ttggggagat tatactttga caaaggtgga aacagcaaat ggatggcaac atttgttcaa 240
tcagctggct tcccagtcgt aatccccttc ttgtttttct tcacaccacc accacccaat 300
tccaccgcca cttcctccca taaaccgcca ccaatctcca ccctcgccct cctctacctc 360
tgctttggcc tactattggc cggtgacaac ttaatgtact cttatggact cctatacctc 420
cctgtctcca cctattcgct cctatgcgca acccaattgg gcttcaatgc agtcttctca 480
ttttttctga attcccaaaa gttcaccgct ctaattctca actccttatt cctcctcacc 540
atctcggcat cgcttcttgc aatccacgca ggctctgata acacgacaaa tgtctccaaa 600
ttgaagtaca caataggctt cctatgcacc ctcggtgcat ctgtcacata ctctttgtac 660
ctctccctaa tccaactctc tttccaacgg gtaatcaaga atgaaacatt tagcgcaatc 720
ttgaatatgc aaatctaccc atcatttgtc gcgacttgtg tttgcattgt ggggcttttt 780
gccagtggag agtggaggac tttgaatggg gagatgaagg tataccaaaa gggaaaggtg 840
tcttatgtga tgactctggt ttggatcgcg gtaacgtggc aaatcgcttc aataggcatg 900
ctcgggctgg tttttgaggt gtcttctctc ttctccaatg tcattggtac tttgggtttg 960
cctgctgttc cgattcttgc tgttgtgttt ttcggcgaca agatggatgg tgtgaaggtg 1020
atatccttgt tgttggctct ctggggcttt ttgtcataca tctatcagca ttatttggat 1080
gactctaaat gtaaagcaac gataacccgt gttgatgtca attctggagc tcggatagag 1140
ctatgtgatc tagagccg 1158
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Phe Phe Leu Leu Ala Gly Gln Ser Ala Ala Val Leu Leu Gly Arg Leu
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Tyr Phe Asp Lys Gly Gly Asn Ser Lys Trp Met Ala Thr Phe Val Gln
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Tyr Ser Leu Leu Cys Ala Thr Gln Leu Gly Phe Asn Ala Val Phe Ser
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Phe Phe Leu Asn Ser Gln Lys Phe Thr Ala Leu Ile Leu Asn Ser Leu
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Phe Leu Leu Thr Ile Ser Ala Ser Leu Leu Ala Ile His Ala Gly Ser
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Asp Asn Thr Thr Asn Val Ser Lys Leu Lys Tyr Thr Ile Gly Phe Leu
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Cys Thr Leu Gly Ala Ser Val Thr Tyr Ser Leu Tyr Leu Ser Leu Ile
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Gln Leu Ser Phe Gln Arg Val Ile Lys Asn Glu Thr Phe Ser Ala Ile
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Leu Asn Met Gln Ile Tyr Pro Ser Phe Val Ala Thr Cys Val Cys Ile
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Ile Ala Val Thr Trp Gln Ile Ala Ser Ile Gly Met Leu Gly Leu Val
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Claims (2)
1.编码茶树咖啡碱转运蛋白CsPUP10.1的基因在提高酵母咖啡碱耐受性中的应用，所述基因的核苷酸序列如SEQ ID No.1 所示。
2.一种制备咖啡碱耐受性提高的转基因酵母的方法，其特征在于包括如下步骤：将编码基因导入酵母中，使该基因过量表达，以减轻咖啡碱对细胞的毒害作用，增强酵母对咖啡碱的耐受能力，所述编码基因的核苷酸序列如SEQ ID No.1 所示。
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