小麦、大麦K~+电压门控通道蛋白β亚基基因分子克隆及序列分析
摘要
采用PCR扩增克隆法,首次从普通小麦(良麦4号和中国春)、野生二粒小麦和大麦中分离得到4个编码钾离子通道蛋白β亚基的基因序列,从分子水平上研究β亚基基因在拟南芥、水稻、小麦、大麦中的遗传变异和氨基酸序列差异,并进行了生物信息学预测、分析,主要研究结果如下：
1.来自良麦4号、中国春、野生二粒小麦与大麦的钾离子通道蛋白β亚基的基因均为987bp,编码328个氨基酸残基的蛋白质序列。将克隆得到的基因序列与已知的K+通道蛋白β亚基基因序列KAB1和KOB1进行比对,同源性分别达到92.08%和95.08%,克隆得到的基因序列具有完整的开放阅读框架,具有起始密码子ATG和终止密码子TAA。与KAB1和KOB1的氨基酸序列比对同源性分别达到96.04%和95.37%。据此认为,克隆得到的基因序列是编码小麦、大麦钾离子通道蛋白β亚基的基因,分别命名为KTB1和KHB1。
2.在KOB1中,第79位到第115位的氨基酸与KAB1中同位置的氨基酸存在很大差异,有34个氨基酸发生了改变其中包括一个缺失。而KTB1和KHB1在该位置与KAB1是高度同源的,仅有2个位点发生了变异,分别是脯氨酸→谷氨酰胺,苏氨酸→亮氨酸之间的替换。在中国春中,该区域第115位酪氨酸→半胱氨酸的替换导致其缺少了1个酪氨酸激酶磷酸化位点。bovKvβ2和ratKvβ1在该区域也相当保守,仅有1个位点的差异,即谷氨酸→谷氨酰胺的替换。KOB1在该区域缺少了3个蛋白激酶C磷酸化位点(STK、SRK、SLK)和1个酪氨酸激酶磷酸化位点(KRLDM (D/E) Y)而多出1个酪蛋白激酶Ⅱ磷酸化位点(SSWE),而植物和哺乳动物仅有的4个共同蛋白激酶C磷酸化位点在该区域出现了2个(S/TTK、SRK)。
3.生物信息学分析表明,KTB1和KHB1没有信号肽,是一个没有跨膜区域的亲水性蛋白。对其二级结构进行预测分析发现,该蛋白无规则卷曲(Coil, c)含量最高,其次是α螺旋(Helix, h),分别为46.65%和39.02%,而β折叠(Strand, e)含量最低,为14.33%。亚细胞定位结果表明该蛋白主要存在于细胞质(60.9%)中,其次是线粒体(26.1%),然后是细胞核(8.7%),极少部分存在于细胞骨架中(4.3%)。
Based on PCR cloning, four gene sequences encoding voltage-gated K+ channelβsubunit were isolated from wheat (cv Liang mai 4, Chinese spring), wild emmer and barley for the first time. Amino acids sequences variations ofβsubunit in Arabidopsis, rice, wheat and barley were detected. Bioinformatics analysis was also conducted. The results were outlined as following.
1. The voltage-gated K+ channelβsubunit sequences from Liang mai 4, Chinese spring, wild emmer and barley were 987bp, encoding 328 amino acids. The nucleotide sequences of KTB and KHB shared 92.08% and 95.08% sequence similarity with those from Arabidopsis and rice K+ channelβsubunits(KAB1 and KOB1), respectively. The genes had complete open reading frames, start codon ATG, and stop codon TAA. The amino acids sequences shared 96.04% and 95.37% sequence identity with KAB1 and KOB1. These clones, the first K+ channel from wheat and barley, were designated as 'KTB1' and 'KHB1'.
2. The region of the KOB1 sequence (amino acids 79-115) shared little homology with KAB1,34 amino acids were mutated including a deletion. However, KTB1 and KHB1 had a high degree of sequence conservation in this region for each other; only 2 mutations, which were Pro→Gln and Thr→Leu, were found. In Chinese Spring, the mutation of Tyr115→Cys115 in this region resulted in a deletion of tyrosine kinase phosphorylation site. KOB1 lacked 3 protein kinase C phosphorylation sites (STK, SRK, SLK) and 1 tyrosine kinase phosphorylation site but had an additional casein kinaseⅡphosphorylation site (SSWE) in this region. Two out of 4 protein kinase C phosphorylation sites which both plant and mammalian shared are present here (S/TTK, SRK).
3. Bioinformatics analysis indicated that KTB1 and KHB1 were hydrophilic polypeptides without signal peptides or membrane spanning regions. Secondary structure prediction showed that they had more coil (46.65%) andα-helix (39.02%) thanβ-strand(14.33%). Subcellular location prediction demonstrated that KTB1 and KHB1 mainly existed in cytoplasm (60.9%), followed by chondriosome (26.1%) and nuclear (8.7%), the rest (4.3%) were located in cytoskeletal.
1.来自良麦4号、中国春、野生二粒小麦与大麦的钾离子通道蛋白β亚基的基因均为987bp,编码328个氨基酸残基的蛋白质序列。将克隆得到的基因序列与已知的K+通道蛋白β亚基基因序列KAB1和KOB1进行比对,同源性分别达到92.08%和95.08%,克隆得到的基因序列具有完整的开放阅读框架,具有起始密码子ATG和终止密码子TAA。与KAB1和KOB1的氨基酸序列比对同源性分别达到96.04%和95.37%。据此认为,克隆得到的基因序列是编码小麦、大麦钾离子通道蛋白β亚基的基因,分别命名为KTB1和KHB1。
2.在KOB1中,第79位到第115位的氨基酸与KAB1中同位置的氨基酸存在很大差异,有34个氨基酸发生了改变其中包括一个缺失。而KTB1和KHB1在该位置与KAB1是高度同源的,仅有2个位点发生了变异,分别是脯氨酸→谷氨酰胺,苏氨酸→亮氨酸之间的替换。在中国春中,该区域第115位酪氨酸→半胱氨酸的替换导致其缺少了1个酪氨酸激酶磷酸化位点。bovKvβ2和ratKvβ1在该区域也相当保守,仅有1个位点的差异,即谷氨酸→谷氨酰胺的替换。KOB1在该区域缺少了3个蛋白激酶C磷酸化位点(STK、SRK、SLK)和1个酪氨酸激酶磷酸化位点(KRLDM (D/E) Y)而多出1个酪蛋白激酶Ⅱ磷酸化位点(SSWE),而植物和哺乳动物仅有的4个共同蛋白激酶C磷酸化位点在该区域出现了2个(S/TTK、SRK)。
3.生物信息学分析表明,KTB1和KHB1没有信号肽,是一个没有跨膜区域的亲水性蛋白。对其二级结构进行预测分析发现,该蛋白无规则卷曲(Coil, c)含量最高,其次是α螺旋(Helix, h),分别为46.65%和39.02%,而β折叠(Strand, e)含量最低,为14.33%。亚细胞定位结果表明该蛋白主要存在于细胞质(60.9%)中,其次是线粒体(26.1%),然后是细胞核(8.7%),极少部分存在于细胞骨架中(4.3%)。
Based on PCR cloning, four gene sequences encoding voltage-gated K+ channelβsubunit were isolated from wheat (cv Liang mai 4, Chinese spring), wild emmer and barley for the first time. Amino acids sequences variations ofβsubunit in Arabidopsis, rice, wheat and barley were detected. Bioinformatics analysis was also conducted. The results were outlined as following.
1. The voltage-gated K+ channelβsubunit sequences from Liang mai 4, Chinese spring, wild emmer and barley were 987bp, encoding 328 amino acids. The nucleotide sequences of KTB and KHB shared 92.08% and 95.08% sequence similarity with those from Arabidopsis and rice K+ channelβsubunits(KAB1 and KOB1), respectively. The genes had complete open reading frames, start codon ATG, and stop codon TAA. The amino acids sequences shared 96.04% and 95.37% sequence identity with KAB1 and KOB1. These clones, the first K+ channel from wheat and barley, were designated as 'KTB1' and 'KHB1'.
2. The region of the KOB1 sequence (amino acids 79-115) shared little homology with KAB1,34 amino acids were mutated including a deletion. However, KTB1 and KHB1 had a high degree of sequence conservation in this region for each other; only 2 mutations, which were Pro→Gln and Thr→Leu, were found. In Chinese Spring, the mutation of Tyr115→Cys115 in this region resulted in a deletion of tyrosine kinase phosphorylation site. KOB1 lacked 3 protein kinase C phosphorylation sites (STK, SRK, SLK) and 1 tyrosine kinase phosphorylation site but had an additional casein kinaseⅡphosphorylation site (SSWE) in this region. Two out of 4 protein kinase C phosphorylation sites which both plant and mammalian shared are present here (S/TTK, SRK).
3. Bioinformatics analysis indicated that KTB1 and KHB1 were hydrophilic polypeptides without signal peptides or membrane spanning regions. Secondary structure prediction showed that they had more coil (46.65%) andα-helix (39.02%) thanβ-strand(14.33%). Subcellular location prediction demonstrated that KTB1 and KHB1 mainly existed in cytoplasm (60.9%), followed by chondriosome (26.1%) and nuclear (8.7%), the rest (4.3%) were located in cytoskeletal.
引文
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7.赵景云,赵宏伟.钾素用量对春玉米及产量性状的影响.东北农业大学学报,2009,40(8)：10-13.
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9.王桂良,黄玉芳,叶优良.不同钾肥品种和用量对小麦产量品质和养分吸收利用的影响.干旱地区农业研究,2009,27(5)：35-40.
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2.刘全喜,马连运.衡水耕层土壤钾素状况及施钾效果[J].土壤肥料,1997,4(1)：29-31.
3.胡思农,涂仕华.四川省作物钾素营养和钾肥应用研究[M].成都：四川科学技术出版社,1999：108-109,108.
4.严蔚东,王校常,何锶洁等.利用外源钾通道基因改良水稻钾素营养.中国水稻科学,2002,16(1)：77-79.
5.许海涛,班新河,许波.钾肥施用对玉米干物质生产及籽粒产量影响研究[J].中国土壤与肥料,2009,(3)：48-50.
6.林咸永,何念祖,章永松等.不同水稻品种对钾的吸收和利用的差异及其与产量和品质的关系.土壤通报,1995,26(7)：49-52.
7.赵景云,赵宏伟.钾素用量对春玉米及产量性状的影响.东北农业大学学报,2009,40(8)：10-13.
8.孙斌.不同钾素水平下小麦纹枯病发生与小麦参量形成因素的关系[J].江苏农业科学,2009,4(3)：133-135.
9.王桂良,黄玉芳,叶优良.不同钾肥品种和用量对小麦产量品质和养分吸收利用的影响.干旱地区农业研究,2009,27(5)：35-40.
10.孙斌,曹雯梅,王应君等.不同土壤钾素对强筋小麦产量形成因子的影响.中国农学通报,2009,25(16)：146-149.
11.李国怀,刘娟旭.施钾肥对温州蜜柑果实发育、裂果和品质的影响.果树科学.1999,16(1)：43-46.
12.印莉萍,黄勤妮,吴平.植物营养分子生物学及信号转导[M].北京：科学出版社,2006：110-111,111.
13.周伯瑜,杨子江.植物缺钾诊断与施钾[J].生命世界,1993,1(1)：42-43.
14. Anderson JA,Huprikar SS,Kochian LV,et al.Functional expression of probable potassium channel Arabidopsis thaliana in S.cerevisie[J].Proc.Natl.Acid.Sci.USA,1992,89(9):3736-3740.
15. Sentenac H,Bonneaud N,Minet M,et al.Cloning and expression in yeast of a plant potassium ion transport system[J].Science,1992,256(5057):663-665.
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