Molecular cloning and expression analysis for PbPR5 on Pyrus bretschneideri ‘Huangguan'

Abstract:【Objective】Plants incur complex cellular,physiological,and molecular responses during theirdevelopmental processes. A group of plant- coded proteins induced by different stress stimuli,named“Pathogenesis-related proteins”(PRs) are given an important role in plant defense against pathogenicconstraints and also in their general adaptation to a stressful environment. A large body of experimentaldata has been accumulated and it provides changing views and concepts on this hot topic as to how it hasevolved. However,until now,there has been no gene identification and expression analysis reportedwhich is related to the pear. In our current study,we isolated the thaumatin-like protein Pb PR5 genefrom Pyrus bretschneideri‘Huangguan'and its expression patterns were analyzed for different tissues,dif-ferent stages of the leaves and fruits and under various environmental conditions.【Methods】Tissue cul-ture seedlings of‘Huangguan'pear were used to study gene expression levels in all of our environmen-tal stress experiments. Seedlings were maintained according to the following procedures: pear seedlingswere maintained in a 1/2MS+0.3 mg·L-1IBA liquid medium under a 16∶8 h light:dark photoperiod(cool white fluorescent tubes,40-60 μmol·m- 2·s- 1) at 25 ℃ in the culture room. All tissues with roots weregrown in 1/2MS+0.3 mg·L-1IBA liquid medium for 2 weeks at 25 ℃ with a 16∶8 h light:dark photoperiod(cool white fluorescent tubes,40-60 μmol·m-2·s-1). To mimic salt- and drought-induced stress,1/2MS+0.3 mg·L-1IBA liquid medium with 150 mmol·L^-1 Na Cl or 20% PEG6000(m/V) were employed to main-tain rooted seedling growth. Seedlings in 1/2MS+0.3 mg·L^-1 IBA liquid medium were used as a control.After 0,6,12,24,48 and 72 h,young roots,stems and leaves from three seedlings were collected. Forthe SA treatment process,the intact plantlets were treated for 72 h by 0.1 mmol·L^{- 1}SA. For ethylenetreatment,tissue culture seedlings were treated with 10 μL·L^-1 ethephon. After 72 h,three seedlingswere collected,then flash frozen in liquid nitrogen,and stored at- 80 ℃ for further analysis. For gene ex-pression in fruits,they were collected from 12 years old pear trees during four,eight and sixteen weeksafter anthesis. Ten trees were selected at random and two fruits were used to isolate the RNA. For gene ex-pression during different stages of leaf development,four branches were selected from the north,south,east,and west positions,and the fifth leaf from the top to the bottom of the branches was recorded duringApril. Leaves were also collected during May,July and September and then flash frozen in liquid nitro-gen,and stored at-80 ℃ for further analysis. For gene cloning,tissue culture seedlings were treated with0.1 mmol·L^-1 SA. Leaves were harvested after 6,12,24,48 and 72 h and pooled. Total DNA was extractedfrom the pooled pear leaves. The nucleotide sequence of the full-length c DNA was determined by RT-PCR. Identification of ORFs in the nucleotide sequence and translation of the deduced amino acid se-quences were completed using the Bio XM 2.6. The genomic structure organization was investigated usinga Gene Structure Display Server(http://gsds.cbi.pku.edu.cn). Homology searches of the NCBI(NationalCenter for Biotechnology Information) databases were performed using BLAST(http://www.ncbi.nlm.nih.gov/BLAST) with default parameters. Multiple alignments of deduced amino acid sequences were carriedout using DNAMAN software with default parameters. Gene expression analysis was performed usingq RT-PCR. 【Results】The c DNA sequence length was 741 bp with a 675 bp open reading frame,encod-ing showed a deduced protein of 224 amino acids. The length of genomic DNA of Pb PR5 was 815 bp withan intron. The results showed the homology of amino acids sequence of Pb PR5 with other PR5 genes from Malus pumila,Prunus mume,Prunus persica,Populus euphratica,Theobroma cacao and Fragaria vesca sub. sp. Vesca was higher,and recorded separately at 96%,84%,85%,83%,79% and 78% respectively.The results of the q RT-PCR showed that the transcript level of Pb PR5 was the highest in the roots,nextin the stem and was lowest in the leaf. The expression of Pb PR5 was induced by salt and drought stresses,and the peak was obtained in the roots in 6 h after Na Cl and PEG6000 treatments,and in leaves in 24 and 12 h,respectively. In addition,the expression was also induced by SA and ethylene. During the dif-ferent development stages of the leaves,the expression level was the highest in the old leaves,next in themature leaves and no expression was noted in the young leaves. The transcript abundance was the highestin the mature fruit than that in the young and expanded fruit.【Conclusion】Taken together,the amino ac-id encoding of the thaumatin-like protein is conserved among the species. The expression level of Pb PR5 was different in the root,stem and leaf for the tissue culture seedling of Pyrus bretschneideri Rehd.‘Huangguan'. Pb PR5 m RNA accumulation was different among the various development stages of theleaf and fruit. Pb PR5 m RNA accumulation in old leaves and mature fruits was the highest. The results sug-gest that the Pb PR5 gene was involved in regulating the mature and senescence processes of leaves andfruits. That salt stress,drought stress and hormone treatment stimulated Pb PR5 expression suggests that Pb PR5 exerts an important role in the responses of pear to unfavorable environmental conditions.