中国农业科学 ›› 2019, Vol. 52 ›› Issue (7): 1119-1135.doi: 10.3864/j.issn.0578-1752.2019.07.001
所属专题: 葡萄研究专题
梁国平,李文芳,陈佰鸿,左存武,马丽娟,何红红,万鹏,安泽山,毛娟()
收稿日期:
2018-07-25
接受日期:
2018-10-15
出版日期:
2019-04-01
发布日期:
2019-04-04
通讯作者:
毛娟
作者简介:
梁国平,Tel:18298344227;E-mail: 基金资助:
LIANG GuoPing,LI WenFang,CHEN BaiHong,ZUO CunWu,MA LiJuan,HE HongHong,WAN Peng,AN ZeShan,MAO Juan()
Received:
2018-07-25
Accepted:
2018-10-15
Online:
2019-04-01
Published:
2019-04-04
Contact:
Juan MAO
摘要:
【目的】探究不同外源糖对葡萄试管苗生长发育及蛋白激酶基因转录调控的影响,应用转录组测序挖掘蛋白质磷酸化过程中的基因,为葡萄蛋白激酶相关基因功能的验证奠定一定基础。【方法】在基本培养基中分别添加2%的蔗糖、葡萄糖和果糖,以无糖为对照,分别命名为S20、G20、F20和CK,经过37 d培养后,测定不同处理的地上和地下鲜重,并采用Illumina HiSeq TM 2000对各处理叶片进行转录组测序,通过综合生物信息学分析(参考基因组比对、差异基因(DEGs)筛选、COG(Cluster of Orthologous Groups of proteins)注释、GO(Gene Ontology)注释等)筛选出蛋白激酶相关基因,通过qRT-PCR分析该蛋白激酶相关基因的表达特性。【结果】F20、G20和S20处理下的葡萄(‘红地球’)试管苗与CK相比,地上鲜重具有明显差异,且F20最高,而G20地下鲜重最高。SNP统计发现,转换是主要的变异类型,颠换次之,且发生在基因间区的SNP数量最多,其次是下游;剪接位点供体和同义终止发生的基因数量最少且相等。4个样品中共获得了2 633个差异基因,3个处理与CK相比,共有差异基因180个且被聚类为3组,第一组中127个基因仅在CK中高表达,第二组19个基因仅在G20下高表达,而第三组34个基因在3个处理下表达模式不尽相同。这些共有的差异基因在COG中注释到了26个基因并分在11个功能类别中,且主要注释在一般的功能类别中。在GO分类中,共有的基因分别被注释在分子功能、生物学过程和细胞组分的14、22和13个功能类别中。共筛选出7种蛋白激酶,分别为葡萄糖激酶(Glucokinase,GK)、丝裂原活化蛋白激酶(Mitogen-activated protein kinases,MAPKs)、钙调蛋白激酶(Calcineurin protein kinase,CBL)、蛋白磷酸酶2(Protein phosphatase 2,PP2A)、己糖激酶(Hexokinase,HXK)、组氨酸蛋白激酶(Histidine protein kinase,HPK)和酪氨酸激酶(Tyrosine kinase,TK),其不同激酶的基因在不同处理中具有各自的表达模式,经qRT-PCR验证,选择的20个差异基因中有17个基因表达与转录组测序结果相一致。【结论】在葡萄试管苗培养中,果糖较葡萄糖和蔗糖相比对生长较好。测序得出180个差异基因对3种不同糖均作出响应,这些基因在COG数据库中主要富集在膜酯转运和代谢、次级代谢物和碳水化合物的合成、转运和分解;GO中大多注释在蛋白激酶和氧化还原酶的活性中;筛选出了7种蛋白激酶,这些差异基因在数量、功能分类和代谢通路上对糖的响应各不相同。
梁国平,李文芳,陈佰鸿,左存武,马丽娟,何红红,万鹏,安泽山,毛娟. 不同糖源对葡萄试管苗蛋白激酶相关基因表达的影响[J]. 中国农业科学, 2019, 52(7): 1119-1135.
LIANG GuoPing,LI WenFang,CHEN BaiHong,ZUO CunWu,MA LiJuan,HE HongHong,WAN Peng,AN ZeShan,MAO Juan. Effects of Different Sugar Sources on Protein Kinase Gene Expression in Grape Plantlets[J]. Scientia Agricultura Sinica, 2019, 52(7): 1119-1135.
表1
qRT-PCR引物"
基因号 Gene ID | 引物序列(5′-3′) Primer sequence (5′-3′) | |
---|---|---|
GSVIVG01014744001 | F-TCAAGGACATCACCACCACA | R-TAGGCCCTTTACGACACACA |
GSVIVG01015297001 | F-ATAGAAGACGCGGTTGGACA | R-TACCCAAGATCACTGCAGCA |
GSVIVG01014081001 | F-AAGACAAGCAGCCATCTCCT | R-TGTGCCTGCAACAGCTTTAG |
GSVIVG01019739001 | F-AGGATTGGTTGAAGGCTCCA | R-AAGGCGTGACTCAGATGGAT |
GSVIVG01026984001 | F-TTGAAGGGGCACTGTCTCAT | R-ACTGGTGGGTCTGGATTGAA |
GSVIVG01038192001 | F-AGCCTCCAATCATGCCCATA | R-ATGATCCATGTGCCGCAAAA |
GSVIVG01038760001 | F-ACGTTAGTGGAGGGAATGCT | R-TGTCCCAATGTTCTCCCCAT |
GSVIVG01001347001 | F-TGGCCCTGAAAGTCCGTTAT | R-TTCAACCAAGGCCGTCAATG |
GSVIVG01020128001 | F-GGAAGCAGCAATAACGTGGT | R-TCTGCCAAGAGACAACCCAA |
GSVIVG01025420001 | F-GGGTTGGGTGCTGTTTTCAA | R-TGCCTCCTTATGCCGAAACT |
GSVIVG01038559001 | F-GGGGTCCAAATTCTTTCGCA | R-TTCCTCGCCATCATCATCCA |
GSVIVG01000919001 | F-TTCGGGCAAGTTTTGGAAGG | R-AAAAGGGAGAAGGAGGTGGA |
GSVIVG01011043001 | F-GGGTTGGACGGCTGAATTTT | R-GGAGGAACGAAACAATGGCA |
GSVIVG01020071001 | F-ATGAGGTTGGCTGCTACTGA | R-AAGGCAAGAACACTGTCCCT |
GSVIVG01000561001 | F-TCTTGGGCTTGGATGGAGTT | R-AGCATGTCGGTCCACTCTTT |
GSVIVG01007646001 | F-AGGCATGGAGAGAAGAAGCA | R-TCTGGGAGGTTTTCAGCACT |
GSVIVG01009192001 | F-AGCGAGGGTGTAAGGTTTGT | R-TCAGCAGTCACATCCTTGGT |
GSVIVG01009685001 | F-ACGGAACTCAGCGTATCAGT | R-TCGTCATCATCGGGTTTCCA |
GSVIVG01013564001 | F-GTTCCGGCCAAATATGAGCA | R-TTGACCGGAAGAAATTCGCC |
GSVIVG01014110001 | F-TGTAGCTGCACCAACAACAC | R-TGGAGGGATTGAACCGTTGA |
GSVIVG01014128001 | F-AGGCTGCCACAAACAACTTT | R-TGCGGATGTTGTAGAGCAGA |
GSVIVG01023376001 | F-TGGACTGTGAGGCTAAACGT | R-TTTAAAAGCCTAGCCGTGCC |
GSVIVG01023676001 | F-TCATCATCCTCATCGCCATCA | R-AACTTGTCGCATGGTTGGTC |
GSVIVG01026000001 | F-AGCGTTTGGGGACTTGAGTA | R-TCCCAGAGCAAGCAAGTACA |
GSVIVG01026487001 | F-TGACGGGAAAGCAGGAAGAA | R-AAACATGCCCAAAAGTCCCC |
GSVIVG01029835001 | F-TGGCTTACGGAGGTGAACTT | R-AGCACCCAGAGCAATCATCA |
GSVIVG01032611001 | F-TTCCCCACCCATCATTCACA | R-TACGGTCATGGTGTAGGCAA |
GAPDH | F-TTCTCGTTGAGGGCTATTCCA | R-CCACAGACTTCATCGGTGACA |
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