Cloning and Expression Analysis of Two Neutral/alkaline Invertase Gene in Garlic

doi:10.7668/hbnxb.20193859

Abstract

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

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.

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Figures/Tables 15

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

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

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

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

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.

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

Fig.6 Conserved motif analysis of AsNI proteins

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

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).

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

Fig.10 Protein interaction network of garlic AsNI1 and AsNI2

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

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

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

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

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