CN116694647B - 猕猴桃盐胁迫响应基因AvMYB08及其应用 - Google Patents
猕猴桃盐胁迫响应基因AvMYB08及其应用 Download PDFInfo
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Abstract
本发明提供了一种序列为SEQ ID NO.1的猕猴桃盐胁迫响应基因AvMYB08及其在耐盐猕猴桃品种育种中的应用。实验表明过表达该基因可以有效提高植物的耐盐性能，提高植物的自由基清除能力，导致氧化胁迫响应基因AtOZF1与脯氨酸合成标记基因AtP5CS1的转录水平上调；为培育耐盐植物，特别是耐盐猕猴桃品种提供了有效的工具。
Description
技术领域
本发明属于基因工程领域，具体地，本发明提供了一种猕猴桃盐胁迫响应基因AvMYB08及其应用。
背景技术
盐胁迫影响植物生长发育过程，包括种子萌发、营养生长和生殖生长。盐渍化土壤中高浓度的钠限制了植物对水和营养物质的吸收。水分缺乏和营养匮缺首先导致细胞离子平衡和渗透压的破坏和其它初级损伤，然后产生大量的活性氧，导致次级损伤如氧化胁迫。离子毒害和渗透胁迫会导致植物生长迟缓及光合作用减弱，进一步的氧化损伤会导致细胞膜结构被破坏甚至引起程序性死亡，导致作物产量减少和死亡。因此，盐胁迫已成为全球农业可持续发展的主要障碍。目前常用的土壤改良方法费时费力，且易导致土壤硬化，难以达到预期的效果。提高生产力和产量，确保全球水果安全的关键策略是培育耐盐品种，筛选耐盐砧木和培育适合当地生长的野生品种，以提高植物的耐盐能力。为了实现这一目标，有必要了解盐渍化土壤对植物形态、生理、生化、代谢和基因表达特征的影响。
猕猴桃根系肉质，偏好中酸性土壤，大多分布在距地面40厘米左右的上层土壤，而这一深度正好是盐分积累和沉积的区域。对盐胁迫较为敏感，当土壤含盐量达到0.14％时，会造成盐害，影响果树的正常生长。当土壤盐分含量达到0.54％时，产量急剧下降，甚至导致植株死亡。一般来说，高盐环境下果树的盐害是由高浓度Na+引起的毒害作用，当果树的细胞摄入超过一定浓度时，会改变大分子的结构、生物膜的结构和功能，通过交换或与大分子结合，降低各种酶的活性，造成盐害。土壤中的大量盐分促进了土壤溶液浓度的增加，从而降低了根际水势，植物吸水能力下降，导致严重的水分流失，水分匮缺导致叶片气孔闭合，从而影响植物的光合作用，最终表现为生理性干旱现象。离子胁迫和渗透胁迫会导致代谢紊乱，产生大量的活性氧，并引起氧化胁迫。活性氧增加细胞膜的通透性，使有机物和离子渗透进入细胞，破坏细胞内的原有平衡，从而影响细胞的功能。过量的活性氧还会导致蛋白质功能失常，酶失活、蛋白质降解和脂质过氧化。此外，活性氧还会直接损伤植物的叶绿体和线粒体，影响光合作用和呼吸作用中的电子转运效率，从而破坏植物的光合作用和呼吸作用，导致DNA构象的改变。猕猴桃在盐胁迫下的主要表现是有机物积累减少、营养物质供应不足、茎干和节间缩短、生长受到抑制。
当植物受到盐胁迫时，膜会触发钙信号、SOS通路和激素传递。接收到信号后，转录因子通过对下游靶基因的精确调控参与耐盐调控网络。转录因子通过激活或抑制一个或多个基因的特异性表达，进一步控制下游基因或直接保护基因的表达，减少盐胁迫引起的损伤。MYB转录因子家族是植物中最大的转录因子家族之一，其中主要R2R3型MYB转录因子涉及到盐胁迫响应。
发明内容
本发明获取了AvMYB08基因，并通过生物信息学分析结合分子生物学试验的方式研究AvMYB08在盐胁迫响应中的功能，为揭示猕猴桃的耐盐分子机理提供理论依据并为耐盐猕猴桃的培育提供了新的途径。
一方面，本申请提供了猕猴桃盐胁迫响应基因AvMYB08，其序列为SEQ IDNO.1。
另一方面，本申请提供了猕猴桃盐胁迫响应蛋白，其序列为SEQ ID NO.2。
另一方面，本申请提供了上述基因或蛋白在提高猕猴桃耐盐性能中的应用。
另一方面，本申请提供了上述基因或蛋白在培育耐盐猕猴桃品种中的应用。
另一方面，本申请提供了上述基因或蛋白在提高猕猴桃自由基清除能力中的应用。
上述基因或蛋白会导致氧化胁迫响应基因AtOZF1与脯氨酸合成标记基因AtP5CS1的转录水平上调。
进一步地，所述应用中过表达上述基因或者施用上述蛋白。
进一步地，所述应用中使用pCAMBIA3301载体。
另一方面，本申请提供了检测上述基因或蛋白的试剂在在耐盐猕猴桃品种育种中的应用。
进一步地，所述应用中检测上述基因表达水平或者蛋白水平，并选用上述基因表达水平或者蛋白水平高的品种用于育种。
进一步地，所述应用中使用序列为SEQ ID NO.1和SEQ ID NO.2的引物进行检测。
检测基因表达水平或蛋白水平的方法和试剂本领域技术人员公知，包括但不不限于各种基于PCR的检测方法和基于抗原—抗体反应的检测方法。
附图说明
图1为以拟南芥为参考，对AvMYB家族进行系统发育分析结果图；
图2为AvMYB家族的多序列比对结果图；
图3为AvMYB家族表达量分析；其中的I,II,III和IV代表盐处理后0，12，24和74h；
图4为盐胁迫响应基因功能预测；
图5为A为AvMYB08亚细胞定位；B为AvMYB08的CDS和蛋白序列
图6为转基因拟南芥与对照对盐胁迫的响应：(A)转基因和对照的qRT-PCR结果；(B)转基因和对照系的耐盐表型；(C)盐胁迫后转基因和对照的存活率；(D)转基因和对照在正常条件与盐胁迫条件下的总黄酮含量；(E)转基因和对照在正常条件与盐胁迫条件下的Fv/Fm图像；(F)转基因和对照在正常条件与盐胁迫条件下的Fv/Fm值。不同字母代表差异显着(p<0.05)。
图7为AvMYB08在盐胁迫下的抗氧化能力：(A)NBT染色；(B)DAB染色；(C)台盼蓝染色；(D)O2 －含量；(E)H2O2含量；不同字母代表差异显着(p<0.05)。
图8为转基因和对照系在正常条件与盐胁迫条件下的生理指标分析：(A)CAT活性；(B)SOD活性；(C)POD活性；(D)Proline含量；(E)MDA含量；(F)电解质渗出率；不同字母代表差异显着(p<0.05)。
具体实施方式
为了使本发明所解决的技术问题、技术方案及有益效果更加清楚，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明；本发明的保护范围仅由权利要求限定。
实施例1AvMYB08的家族分析和功能预测
发明人从‘对萼’猕猴桃全长转录组数据中筛选出AvMYB家族成员，去除重复和不完整的序列。以‘红阳’猕猴桃为参考，筛选具有MYB保守结构域(PF00249)的成员，最终获得44个MYB基因。
发明人对AvMYB家族进行了系统发育分析，以拟南芥为参照，进行了适当的调整，并将其分为6个亚家族(图1)。结果表明，AvMYB08属于亚家族III。已报道的在拟南芥受中响应盐胁迫的MYB12，AtMYB20和AtMYB111也属于这个亚家族，特别是AtMYB111与AvMYB08聚在了同一个分支，可能是直系同源基因并具有相似的功能。MYB通常分为四类:4R-MYB、R2R3-MYB、R1R2R3-MYB(3R-MYB)和MYB-related蛋白，其中R2R3型MYB基因通常响应胁迫反应，多序列比对分析结果表明AvMYB08属于R2R3型MYB基因(图2)。
表达模式结果表明，在盐胁迫后，亚家族I的AvMYB11、AvMYB15、AvMYB24和AvMYB34，亚家族III的AvMYB08，亚家族VI的AvMYB03和AvMYB27的表达明显增加(图3)。基于拟南芥同源基因，发明人采用STRING对盐胁迫响应基因进行了功能预测(图4)。AvMYB03与MYBR1为同源基因，响应脱落酸抑制蛋白磷酸酶2C的表达。在生长素的作用下，AvMYB03通过与PYL8的直接相互作用增强了IAA19的转录活性。AvMYB08与MYB111为同源基因，负责黄酮醇糖苷3的合成，并作为黄酮醇特异性转录激活因子参与黄酮醇生物合成相关基因的调控。激活CHS、CHI、F3H和FLS1的表达，并控制黄酮醇的生物合成。AvMYB11与MYB59为同源基因，功能目前尚不清楚。AvMYB15预测结果与AvMYB11相同。AvMYB24不是R2R3型MYB转录因子。AvMYB27与AS1为同源基因，与细胞分化有关。AvMYB34与MYB79为同源基因，功能目前尚不清楚。在非生物胁迫下，黄酮类化合物可以有效降低ROS对植物细胞的损伤。因此，推测AvMYB08与盐胁迫响应有关。
实施例2AvMYB08的获取和亚细胞定位
发明人以未处理‘对萼’猕猴桃根系反转录合成cDNA第一链为模板，利用引物5'-ATGGGAAGGGCTCCATGTTGT-3'(SEQ ID NO.3)与5'-TCAAATCAACAGAGATTCAGC-3'(SEQ IDNO.4)首次扩增了AvMYB08基因，该基因包含一个完整的825的开放性阅读框，编码274个氨基酸的蛋白，预测分子量31198.31，等电点为9.06。
AvMYB08的基因序列为：
ATGGGAAGGGCTCCATGTTGTTCAAAGGTGGGGTTGCATAGAGGTCCATGGACTGCTAGAGAAGACTCG
TTGCTTACAAACTATATTCAGGTTCATGGTGAAGGCAATTGGAGATCTTTGCCTAAAAAGGCTGGATTG
CTTAGATGTGGAAAGAGTTGCAGACTAAGATGGATGAATTATCTACGACCGGACATCAAGAGGGGGAAT
ATCACCCCAGACGAGGACGACCTTATCGTAAAAATGCATGCCCTTTTAGGCAATCGATGGTCTCTAATC
GCTGGAAGACTACCGGGTCGAACCGATAACGAGATCAAGAATTACTGGAACACCCATCTCACTAAAAAG
CTCCGAAGCCTGGGGACCGACCCGACTACCCACAAAAAGTTATCGGATTCCCACGTACAAGAACAGAAG
AAGAGAAAAAACAGCAGCAAAAAGAAGAAGTCAAAGCCAAAGCCTGAAGCAGAGCCTGAAAAGGTCAAA
GTCCATAATCCAAAGCCCATTAGGATCAAGTCTCTGGGCTCTTTCTCAATGTCAAGAAATACCAGCTTT
GATTGGACGACGACGACGGCTACATCTACAGCTAGTGGTACAGAAGTTGCAGATGATCCATGGTCCAAT
TTCAGATATAATGAGGTTGGTTTTCTGATTGGTGATGATCAAGATCATGATATGATCAACGGCGCAGAT
CTCGAGTGCCAATCTGGTGTACCAATGTCCAATCACACTCTAGAGAAGCTCTATGAGGAGTATTTTCAA
CTGTTGAAGGCAGAAGACCATGACCATGTCGAGTTGGACTCCTTTGCTGAATCTCTGTTGATTTGA(SEQ ID NO.1)
AvMYB08的蛋白序列为：
MGRAPCCSKVGLHRGPWTAREDSLLTNYIQVHGEGNWRSLPKKAGLLRCGKSCRLRWMNYLRPDIKRGN
ITPDEDDLIVKMHALLGNRWSLIAGRLPGRTDNEIKNYWNTHLTKKLRSLGTDPTTHKKLSDSHVQEQK
KRKNSSKKKKSKPKPEAEPEKVKVHNPKPIRIKSLGSFSMSRNTSFDWTTTTATSTASGTEVADDPWSNFRYNEVGFLIGDDQDHDMINGADLECQSGVPMSNHTLEKLYEEYFQLLKAEDHDHVELDSFAESLLI(SEQ IDNO.2)。
通过Xba I和Sal I双酶切构建亚细胞定位载体PB221-GFP-35S-AvMYB08，与空载通过PEG诱导法转入拟南芥原生质体，发现AvMYB08在细胞核和细胞质中均有荧光信号，细胞核中的信号比细胞质中的信号更强(图5)。这可能是因为通过调节表达的转录因子从细胞质到细胞核的易位来进行翻译后调控是快速响应胁迫信号的有效策略。
实施例3AvMYB08在盐胁迫响应过程中的功能验证
为了进一步验证AvMYB08在盐胁迫响应过程中的功能，发明人通过Nco I与BglⅡ双酶切构建过表达载体pCAMBIA3301-35S-MYB08。将pCAMBIA3301-35S-AvMYB08与pCAMBIA3301空载通过热激法转入农杆菌GV3101。蘸花法侵染拟南芥。通过除草剂(1/2000Basta)进行抗性植株筛选，PCR检测T1抗性植株，将筛选出的阳性植株单株种下，收获T2代种子。通过含草铵膦(7mg/L)的MS培养基筛选绿苗与黄苗比例为3:1的T2代阳性植株，将绿苗移入基质，通过草铵膦筛选为转基因纯系后，T3代阳性植株用于后续实验。Fv/Fm图像和Fv/Fm值结果以及表型鉴定表明尽管盐处理后转基因和对照都受到不同程度的损伤，但转基因的表型和存活率明显优于对照，并且含有更多的类黄酮(图6)。
NBT和DAB染色结果与O2 －和H2O2含量检测结果表明AvMYB08能够提高转基因拟南芥抗氧能力。通过台盼蓝对细胞死亡结果进行观察，结果表明AvMYB08在响应盐胁迫时起到积极的作用(图7)。超氧化物歧化酶(SOD)能催化超氧阴离子转化为H2O2和O2，是清除植物体内自由基的重要物质，而过氧化物酶(POD)和过氧化氢酶(CAT)是清除H2O2的酶。SOD、POD和CAT通过协同作用维持植物体内自由基含量的稳定水平，防止自由基引起的植物生理学和生化变化。脯氨酸含量和酶活性结果表明，盐胁迫下转基因植株具有更强的ROS清除能力以及维持渗透平衡的能力。受到盐胁迫后，转基因植株中氧化胁迫响应基因AtOZF1与脯氨酸合成标记基因AtP5CS1的转录水平上调(图8)。上述结果表明，AvMYB08通过抗氧化防御与合成有机渗透物质来保护细胞膜的完整性，从而提高耐盐能力。
Claims (3)
1.猕猴桃盐胁迫响应基因AvMYB08在提高猕猴桃耐盐性能中的应用，所述猕猴桃盐胁迫响应基因AvMYB08的核苷酸序列为SEQ ID NO.1；所述应用中在猕猴桃中过表达所述猕猴桃盐胁迫响应基因AvMYB08。
2.猕猴桃盐胁迫响应基因AvMYB08在培育耐盐猕猴桃品种中的应用，所述猕猴桃盐胁迫响应基因AvMYB08的核苷酸序列为SEQ ID NO.1；所述应用中在猕猴桃中过表达所述猕猴桃盐胁迫响应基因AvMYB08。
3.根据权利要求1或2所述的应用，所述应用中使用pCAMBIA3301载体构建过表达载体来过表达猕猴桃盐胁迫响应基因AvMYB08。
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