Cloning,Subcellular Localization and Expression Analysis of PpLAC7 Gene from Jinxiu Yellow Peach

doi:10.7668/hbnxb.20194985

Abstract

Abstract:

To clone the PpLAC7 gene sequence of Jinxiu yellow peach,investigating its gene sequence information and its role in flesh browning.The cDNA sequence of PpLAC7 was cloned from Jinxiu yellow peach by homologous cloning.The promoter region elements,physicochemical properties,secondary and tertiary structure,phylogenetic tree and amino acid sequence alignment of PpLAC7 gene were performed bioinformatics by using bioinformatics software.In addition,it also investigated the subcellular localization and the expression pattern of PpLAC7 during the process of fresh browning in Jinxiu yellow peach.The results of bioinformatics analysis indicated that the promoter region included light responsiveness,MeJA-responsiveness,drought-inducibility,seed-specific regulation elements,etc.The full length of PpLAC7 gene was 1 692 bp,encoding 563 amino acids,molecular weight was 61.867 ku,total number of atoms was 8 621,GRAVY was -0.028,theoretical pI was 5.95,instability index was 36.48,aliphatic index was 87.26.The secondary structure of PpLAC7 protein mainly contained α-helix of 14.74%,extended strand of 28.77%,β-turn of 6.22% and random coil of 50.27%.Phylogenetic analysis showed that PpLAC7 had high similarity to MdLAC7.Deduced amino acid sequence indicated that PpLAC7 protein,the same as other species,also contained three typical copper ion domains.Subcellular localization analysis revealed that PpLAC7 was located in endoplasmic reticulum.The expression of PpLAC7 gene was up-regulated during the storage of Jinxiu yellow peach fruit.Combining the relationship of flesh browning index and PpLAC7 expression,it indicated that PpLAC7 may play an important role in the browning process of Jinxiu yellow peach fruit.

Key words: Jinxiu yellow peach, PpLAC7 gene, Flesh browning, Gene cloning, Bioinformatics analysis, Subcellular localization

GONG Yihui, ZHANG Canmei, ZHOU Guihua, CHEN Meiyu, TAN Qian, PI Shuiqin, ZENG Yongxian. Cloning,Subcellular Localization and Expression Analysis of PpLAC7 Gene from Jinxiu Yellow Peach[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(5): 72-79. doi: 10.7668/hbnxb.20194985.

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Figures/Tables 10

Tab.1 The primer sequences

基因 Gene	上游引物(5'-3') Forward primer(5'-3')	下游引物(5'-3') Reverse primer(5'-3')	目的 Purpose
PpLAC7	ATGGCACGCTTTGCCTTCGT	CTAACATTGGGGTAGATCCG	基因克隆
PpTEF2	GGTGTGACGATGAAGAGTGATG	TGAAGGAGAGGGAAGGTGAAAG	内参引物
PpLAC7	GGCCCACAAGTTCAACACC	TTACGGGACACAGCTCCAGAT	实时荧光定量 PCR 引物

Fig.1 Ampliation for the cloning of PpLAC7 genes cDNA in Jinxiu yellow peach M.DL2000 DNA Marker;1.The target fragment;2.The control.

Tab.2 Structure of cis-elements in promoter region of PpLAC7 gene

序列 Motif	功能 Function	数量 Number
AAACCA	厌氧诱导的顺式作用调控元件	3
ATTAAT,GGTTAA,GGTTAAT,TGATAATGT,AACCTAA	光响应元件	10
CAAT,CCAAT,CAAAT	顺式作用元件	28
CGTCA,TGACG	茉莉酸响应元件	4
CAACTG	干旱响应元件	1
CATGCATG	种子特异性调控元件	1
TATA,ATATAT,TATATA,TATAAAA	核心启动子元件	28

Fig.2 Secondary structure analysis of PpLAC7 protein in Jinxiu yellow peach Blue, red, green and orange represent alpha helix, extended strand, beta turn and random coil structure, respectively.

Fig.3 Tertiary structure predict of PpLAC7 protein in Jinxiu yellow peach

Fig.4 Phylogenetic trees of PpLAC7 protein in Jinxiu yellow peach

Fig.5 Amino acid sequence alignment analysis of laccase from peach,apple and litchi Ⅰ,Ⅱ and Ⅲ are the types of Ⅰ Cu²⁺, Ⅱ Cu²⁺, and Ⅲ Cu²⁺ of DNA domains, respectively.

Fig.6 Subcellular localization of PpLAC7

Fig.7 Changes of browning index by PE treatment in Jinxiu yellow peach Different capital letters indicate extremely significant difference between different treatments(P<0.01).The same as Fig.8.

Fig.8 Expression analysis of PpLAC7 during the process of flesh browning in Jinxiu yellow peach fruit

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