大蒜2个中性/碱性转化酶基因AsNI的克隆与表达分析

doi:10.7668/hbnxb.20193859

摘要/Abstract

摘要：

中性/碱性转化酶作为植物蔗糖代谢的关键酶,主要参与植物生长发育、逆境胁迫响应等过程。为探究大蒜AsNI对逆境胁迫的响应模式,以乐都紫皮大蒜为试验材料,克隆得到2个中性/碱性转化酶基因,并进行了生物信息学和表达特性分析。结果表明,AsNI1和AsNI2的开放阅读框分别为522,1 203 bp,编码173,400个氨基酸;AsNI1与AsNI2均为亲水性蛋白,预测定位于细胞质,具有Glyco_hydro_100结构域;但二者的氨基酸序列相似性仅为25.75%,AsNI2含有1个糖基化位点,而AsNI1未检测到糖基化位点,二者的亲缘关系较远。蛋白互作网络分析结果表明,AsNI2与AsNI1可能参与不同的生化过程。启动子序列分析发现,AsNI1和AsNI2的启动子区域含有多个与响应相关的顺式作用元件,其中AsNI2启动子的干旱和低温胁迫响应元件个数显著大于AsNI1。通过对启动子转录因子结合位点进行预测发现,二者含有不同种类及数量的结合位点,表明AsNI1和AsNI2可行使不同的基因功能。qRT-PCR检测发现,AsNI的表达具有明显的组织特异性,AsNI1和AsNI2分别在根和鳞茎中表达量最高。同时,逆境胁迫能够诱导AsNI表达,低温和干旱处理下均以AsNI2的响应程度强于AsNI1。其中,低温胁迫以诱导叶片AsNI2的表达为主,干旱胁迫以诱导根系AsNI2的表达为主。通过分析AsNI的序列特征和表达模式,验证了AsNI的抗逆功能。

关键词: 大蒜, 中性/碱性转化酶基因, 基因克隆, 表达分析, 逆境胁迫

Abstract:

Neutral/alkaline invertases,as important substances in plant sucrose metabolism,are mainly involved in processes such as plant growth and development,and response to adversity stress.To investigate the response pattern of AsNI to stress,this study cloned two garlic neutral/alkaline invertase genes,using Ledu purple skin garlic as the test material and they were subjected to bioinformatics and expression characterization.The results showed that the open reading frames of AsNI1 and AsNI2 were 522,1 203 bp,encoded 173,400 amino acids.AsNI1 and AsNI2 were both hydrophilic proteins predicted to be localized in the cytoplasm with a Glyco_hydro_100 structural domain.However,the amino acid sequence similarity between the two was only 25.75%,and AsNI2 contained one glycosylation site,while no glycosylation site was detected in AsNI1,and the two were distantly related.The analysis of the protein interaction network showed that AsNI2 and AsNI1 might participate in different biochemical processes.The promoter sequence analysis revealed that the promoter regions of AsNI1 and AsNI2 contained multiple cis-acting elements related to stress response,with the AsNI2 promoter having a significantly larger number of drought and low temperature stress response elements than AsNI1.The prediction of promoter transcription factor binding sites showed that they contained different kinds and numbers of binding sites,indicated that AsNI1 and AsNI2 could perform different gene functions.The qRT-PCR assay revealed that the expression of AsNI was significantly tissue-specific,with the highest expression of AsNI1 and AsNI2 in the roots and bulbs,respectively.Meanwhile,adversity stress was able to induce AsNI expression,and the response of AsNI2 was stronger than that of AsNI1 under both low temperature and drought treatments.Among them,low temperature stress mainly induced the expression of AsNI2 in leaves,and drought stress mainly induced the expression of AsNI2 in roots.The sequence characteristics and expression pattern of AsNI were analyzed to verify the stress resistance function of AsNI.

Key words: Garlic, Netural/alkaline invertase gene, Gene cloning, Expression analysis, Abiotic stress

周倩怡, 黄思杰, 田洁. 大蒜2个中性/碱性转化酶基因AsNI的克隆与表达分析[J]. 华北农学报, 2023, 38(4): 54-64. doi: 10.7668/hbnxb.20193859.

ZHOU Qianyi, HUANG Sijie, TIAN Jie. Cloning and Expression Analysis of Two Neutral/alkaline Invertase Gene in Garlic[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(4): 54-64. doi: 10.7668/hbnxb.20193859.

http://www.hbnxb.net/CN/Y2023/V38/I4/54

图/表 15

图1 大蒜AsNI基因的RT-PCR扩增图谱 M.DNA分子量标准。

Fig.1 RT-PCR amplication pattern of AsNI gene from garlic M.DNA Marker.

图2 大蒜AsNI1基因的核苷酸序列及其编码的氨基酸序列 *.终止密码子。图3同。

Fig.2 Nucleotide sequences and predicted amino acid sequences of AsNI1 gene from garlic *.Termination codon.The same as Fig.3.

图3 大蒜AsNI2基因的核苷酸序列及其编码的氨基酸序列

Fig.3 Nucleotide sequences and predicted amino acid sequences of AsNI2 gene from garlic

表1 大蒜AsNI的蛋白质理化性质

Tab.1 Physicochemical properties of AsNI protein

理化性质 Physicochemical properties	分子式 Formula	分子量/ku Molecular weight	pI	亲水性平均系数 Grand average of hydropathicity	不稳定系数 Instability index	磷酸化位点 Phosphorylation sites	糖基化位点 Glycosylation site
AsNI1	C₉₁₅H₁₃₇₅N₂₄₁O₂₅₇S₁₂	20 248.16	5.86	-0.368	46.11(不稳定)	18	0
AsNI2	C₂₀₅₄H₃₁₆₂N₅₃₈O₅₉₄S₂₁	45 570.21	5.76	-0.290	41.02(不稳定)	41	1

图4 AsNI1蛋白二级(A)、三级(B)结构预测蓝色.α螺旋;绿色.β转角;黄色.无规则卷曲;红色.延伸结构。图5同。

Fig.4 Secondary(A)and tertiary(B)structure prediction of AsNI1 protein Blue.Alpha helix;Green.Beta turn;Yellow.Random coil;Red.Extended strand.The same as Fig.5.

图5 AsNI2蛋白二级(A)、三级(B)结构预测

Fig.5 Secondary(A)and tertiary(B)structure prediction of AsNI2 protein

图6 AsNI蛋白的保守基序分析

Fig.6 Conserved motif analysis of AsNI proteins

图7 大蒜AsNI1(A)、AsNI2(B)氨基酸序列保守域预测

Fig.7 Prediction of conserved domain in the acid sequences of AsNI1(A), AsNI2 (B) from garlic

图8 AsNI与其他植物NI氨基酸序列的多重比对短柄草(XP_003558048.1∶222-620)、凤梨(OAY70851.1)、柑橘(KAH9692069.1)、高粱(XP_002465359.1∶227-625)、姜(XP_042399050.1∶235-634)、芦笋(XP_020247438.1)、木槿(XP_039002564.1)、拟南芥(KAG7589826.1∶297-469)、铁皮石斛(AVA07409.1∶212-610)、小麦(XP_044375374.1)、野生稻(XP_006645848.2∶240-637)、黑麦草(XP_047072902.1∶223-621)、玉米(XP_008655058.1∶226-624)。

Fig.8 Alignment of the deduced amino acid sequences of AsNI and NI from other plant Brachypodium distachyon(XP_003558048.1∶222-620),Ananas comosus(OAY70851.1), Citrus sinensis(KAH9692069.1),Sorghum bicolor(XP_002465359.1∶227-625), Zingiber officinale(XP_042399050.1∶235-634), Asparagus officinalis(XP_020247438.1), Hibiscus syriacus(XP_039002564.1),Arabidopsis suecica(KAG7589826.1∶297-469), Dendrobium catenatum(AVA07409.1∶212-610),Triticum aestivum(XP_044375374.1), Oryza brachyantha(XP_006645848.2∶240-637), Lolium rigidum(XP_047072902.1∶223-621),Zea mays(XP_008655058.1∶226-624).

图9 大蒜与其他作物NI氨基酸序列的系统进化树

Fig.9 Phylogenetic tree of NI from garlic and other plants

图10 大蒜AsNI1和AsNI2的蛋白互作网络

Fig.10 Protein interaction network of garlic AsNI1 and AsNI2

表2 AsNI基因启动子部分顺式作用元件

Tab.2 Partial cis-acting elements of AsNI gene promoter sequence

元件 Element	序列 Sequence	功能 Function	数量 Amount
元件 Element	序列 Sequence	功能 Function	AsNI1	AsNI2
MYB	CAACCA/TAACCA	MYB 结合位点	7	3
MYC	CATTTG	干旱诱导MYC 结合位点	2	3
CAAT-box	CAAT/CAAAT/TGCCAAC	启动子与增强子区的一般顺式作用元件	22	37
ARE	AAACCA	厌氧诱导所必需的顺式作用的调节元件	2	3
Myb-binding site	CAACAG	MYB结合位点	2	1
TATA-box	TATA/TATAA/ATTATA/	转录起始点-30附近的核心启动子元件	19	29
	TATATA/TATAAAA/TATAAA
MBS	CAACTG	干旱胁迫的MYB响应元件		3
Myb	CAACTG	MYB响应元件		3
LTR	CCGAAA	低温响应元件		1

图11 大蒜AsNI基因不同组织表达分析不同小写字母表示不同处理间差异显著(P<0.05)。

Fig.11 Expression analysis of AsNI gene in different tissues Different lowercase letters indicate significant difference among treatments(P<0.05).

图12 AsNI基因在低温胁迫处理下不同组织相对表达水平

Fig.12 Relative expression levels of AsNI gene in different tissues under low temperature

图13 AsNI基因在干旱胁迫处理下不同组织相对表达水平

Fig.13 Relative expression levels of AsNI gene in different tissues under drought stress

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