Cloning And Expression Of Cytosolic Apx Gene In Embryogenic Cultures Of Dimocarpus Longan Lour

In this study,the main synchronized embryogenic cultures at different somatic embrogenesis stages of longan(Dimocarpus longan Lour.)embryogenic callus(EC)of "Red nucleus" were used for materials as well as longan EC under NaCl,light and temperature stress treatments.Then,researches were performed as follows:①In order to obtain the complete ORF fragment,5′-end sequence of apx gene was cloned by using homology cloning technology on the basis of the known 3′-end sequence;②Cytosolic apx gene was expressed in prokaryotic cells;③The activity changes of APX in longan embryogenic cultures were detected;④With establishment of analytical method of the longan APX isoenzymes by electrophoresis,the changes of APX isoenzymes in longan somatic embryogenesis were analysed well as in longan EC under stress treatments;⑤As cloning the housekeeping gene(β-actin)in longan EC,relative expression of cytosolic apx gene at the level of transcription at different stages of longan somatic embryogenesis and in longan EC under stress treatments were anlayzed by using fluorescence quantitative PCR technology.The main results were as follows:1.The 5′-end sequence cloning and the obtaining of the full length cDNA sequence of cytosolic apx gene from longan ECUsing the longan EC as the material,the 5′-end sequence of cytosolic apx gene was cloned from longan embryogenic callus by 5′RACE.The specific trans-primers were designed according to the known 3′-end sequence and combined with the universal amplification primers to trail-PCR.After primer screening and optimization,a 500 bp specific sequence was obtained, which had 250 bp nucleotide sequence coincided with the known 3'-end sequence.1027 bp sequence could be got by splicing the fragments of the two ends.The results showed the apx gene sequence in longan EC was highly homologous with other plants reported in GenBank.Sequence analysis revealed that the full length cDNA contained a 753 bp open reading frame and encoded 251 amino acids.The deduced amino acid sequence was highly homologous with cAPX in other plants.In addition,there was no intron in the ORF.2.The prokaryotic expression of cytosolic apx gene of longanAccording to the full length cDNA sequences of cytosolic apx gene of longan,we designed a pair of specific primers containing sepcific restriction endonuclease sites at the putative ORF regional.ORF fragment was amplified by PCR and was cloned into the pET-29a expression vector. After being validated,the pET-29a expression vector was transformed into BL21.Being induced,SDS-PAGE analysis showed that a new protein was induced in the host with the molecular weight of approximately 28 kD,which was nearly the same as the calculated molecule weight 27.7 kD of the longan cytosolic APX.3.The changes of APX activity in Iongan EC under NaCl,light and temperature stress treatmentsUnder NaCl,light,and temperature stress treatments,the significant difference of the APX activity in longan EC was detected.The results showed that compared with the control,the APX activity increased at different levels under those stress treatments and its change was related to the resistance of embryonic cells.4.The dynamic changes of APX isoenzymes in the precess of somatic emb -ryogenesi and in longan EC under stress treatmentsIn this study,a simple and efficient method involved extraction,electrophoresis and dying was developed for analyzing of APX isoenzymes from synchronized embryogenic cultures of longan by polyacrylamide gel electrophoresis.Using the method,we analyzed the change of APX isozymes in the processing of longan somatic embryogenesis as well as in the longan EC under stress treatments.The results showed that the dynamic change of APX isoenzymes banded to the differentiation of embryonic cells.There was an obvious difference at different developmental stages.Under stress treatments,dynamic changes of APX isoenzymes related to different stress levels.These results suggested that the response of embryonic cells to oxidative stress did not rely on the function of cAPX alone,but with a variety of APX isoenzymes synergies.Thus,specific mechanisms should be further investigated.5.Expression analysis on cytosolic apx gene of longan at transcription levelIn this assay,the 895 bp 3′-end sequence ofβ-actin gene was cloned from longan EC as the control for cytosolic apx gene expression analysis by real-time quantitative PCR technology. The results showed that comparing to the longan EC,in the process of somatic embryogenesis, the relative gene expression levels increased with the differentiation of embryonic cells.At the stage of compact globular embryoids relative expression levels was highest and then a small drop appeared at the stage of typical globular embryoids and cotyledonary embryoids.Under temperature stress,relative expression of apx gene was higher than that in the control and the effect of high temperature on the change of expression was more obvious.However,the quantity of relative apx gene expression was largest at 40℃.