Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (24): 4906-4915.doi: 10.3864/j.issn.0578-1752.2023.24.009

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Expression Analysis of PtMLP1 Promoter in Poncirus trifoliata

YAO LiXiao(), SU Juan, GUO XingRu, LI FengLong, HE YongRui, ZOU XiuPing, CHEN ShanChun()   

  1. Citrus Research Institute, Southwest University/National Citrus Engineering Technology Research Center/National Center for Citrus Varieties Improvement, Chongqing 400712
  • Received:2023-05-31 Accepted:2023-08-04 Online:2023-12-16 Published:2023-12-21
  • Contact: CHEN ShanChun

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Abstract:

【Objective】Genetic transformation plays a significant role in exploring gene function and improving traits in citrus. Tissue-specific promoters is a key to regulate the expression of transgenes in particular tissues. Here, expression characteristics of the PtMLP1 promoter, isolated from the root subtractive library of Poncirus trifoliata, was thoroughly examined, which could lay a foundation for the specific expression of exogenous genes in citrus root tissue. 【Method】The complete sequence of PtMLP1 gene was cloned by PCR using DNA as a template. The physiochemical attributes, secondary and tertiary structures of PtMLP1 protein were predicted by ExPASy, PSIPRED, and SWISS-MODEL tools. Cis-acting elements in PtMLP1 promoter were predicted by PlantCARE. The expression pattern of PtMLP1 in P. trifoliata trees of diverse ages was examined by employing real-time qPCR. Furthermore, to investigate the tissue-specific expression of the PtMLP1 promoter in citrus, a pBI121-ProPtMLP1::GUS plasmid, in which GUS expression was controlled by the PtMLP1 promoter, was constructed and then introduced into P. trifoliata through Agrobacterium-mediated hypocotyl transformation. 【Result】PtMLP1 consisted of two exons and one intron, which possessed a 471 bp open reading frame encoding a protein with 156 amino acid residues. This protein had a molecular weight of 17.63 kilodaltons with an isoelectric point of 5.49 and contained a Bet v I functional domain in its primary structure. Moreover, the secondary structure of PtMLP1 contained three α-helices and seven β-folds, while its tertiary structure had a conserved hydrophobic binding site and a cyclic domain, which was rich in glycine. The PtMLP1 promoter was 1 666 bp long. Multiple root-specific expression elements, phytohormone response elements (such as the TGACG motif, P-box, and ABRE), and the TATA box and CAAT box core elements were predicted in the promoter. Additionally, the 3-terminal untranslated region of PtMLP1 was predicted to contain a poly (A) signal AATAAA. Notably, the expression of PtMLP1 was significantly higher in the roots of 1-month, 6-month, and 20-year-old P. trifoliata, with fold changes of 46.34, 74.82, and 110.25, respectively, compared with those in leaves. GUS expression analysis of pBI121-ProPtMLP1::GUS transgenic plants showed that PtMLP1 promoter exhibited specific and high expression in roots, and its expression levels were 7.76 to 124.78 times of that in the leaves. 【Conclusion】The sequences of the PtMLP1 gene and its promoter were successfully obtained, and the promoter demonstrated the ability to drive specific expression of GUS gene in citrus roots.

Key words: Poncirus trifoliata, major latex protein, root-specific promoter, GUS

Table 1

The sequence of primers in the experiments"

引物名称
Primer name		 引物序列
Primer sequence		 作用
Function
Con2F 5′-ACCGTTCAAGCCATTCCGAAGG-3′ PtMLP1实时荧光定量PCR
Real time qPCR for PtMLP
Con2R 5′-TGGGGGATGATGGGCTTCGAT-3′
qAct1 5′-CTGCTGGAATTCACGAAACA-3′ Actin实时荧光定量PCR
Real time qPCR for Actin
qAct2 5′-GCCACCACCTTGATCTTCAT-3′
GUSF 5′-CGTTAGCCGGGCTGCACTCA-3′ GUS实时荧光定量PCR
Real time qPCR for GUS
GUSR 5′-CCGCCAACGCGCAATATGCC-3′
2IS 5′-GACCCTATCTGGTCAGTTGGAGAC-3′ 克隆PtMLP1内含子
Cloning the intron of PtMLP1
2IR 5′-CACCCTTCGGAATGGCTTG-3′
2PaS2 5′-TATaagcttATCTTCGGCTGTAGGCCACATCA-3′ PtMLP1启动子克隆
Cloning the promoter of PtMLP1
2PaR1 5′-GCGtctagaGTTTTTAGCGGATTAAAATA-3′

Fig. 1

The DNA sequence of PtMLP1 The black lowercases mean promoter sequence, with the cis-acting elements marked with brown horizontal lines: ① root specific motif, ② TGACT-motif, ③ P-box, ④ CCAAT-box, ⑤ CAT-box, ⑥ ABRE, ⑦ CAAT-box, ⑧TATA-box. The red uppercases mean exons and their encoded amino acids. The green lowercases mean intron sequence. The blue lowercases mean a 3′-terminal untranslated region with the poly (A) signal (AATAAA)"

Fig. 2

The expression of PtMLP1 in different tissues of citrus"

Fig. 3

The Cloning of ProPtMLP1 sequence (A) and schematic diagram of plant expression vector (B)"

Fig. 4

GUS expression in roots and leaves of Poncirus trifoliata transformed with ProPtMLP1::GUS"

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