Cloning Of HbGRX Gene From Hevea Brasiliensis And Functional Analysis Of The HbGRX During The Occurrence Of TPD

Tapping panel dryness (TPD), a complex physiological syndrome of rubber tree (Hevea brasiliensis), is one of the major factors affecting natural rubber production. Using techniques of microscopic anatomy, physiology and molecular biology, bark microscopic anatomy and latex physiological characteristics were analyzed, and a TPD-related gene,HbGRX, was cloned and its expression patterns were investigated, with bark and latex of rubber tree with different severity of TPD as experimental materials. The results of this study provided a basis for further exploring the nature and mechanism of the TPD of Hevea brasiliensis. The main results are as follows:1. On the basis of both the identification of the microscopic characteristics of bark and the determination of the physiological parameters of the latex of rubber trees with different severity of TPD, a TPD classification and grading system was developed for the first time. This provided a basis for the diagnosis and survey of TPD, and the study of the physiological and molecular mechanism of latex during the occurrence of TPD.2. The physiological parameters analysis showed that after the occurrence of TPD,latex production decreased significantly, the content of dry rubber increased, the latex pH decreased significantly, the content of thiols decreased significantly, the content of sucrose increased significantly, lutoid burst index increased significantly and magnesium ions increased significantly. For the first time, this study comprehensively analyzed the changes of various latex physiological parameters of rubber tree of rubber tree with different severity of TPD, which reflected the correlation between the physiological parameters and the occurrence of TPD. This provided a physiological basis for the study the mechanism of TPD,and some physiological parameters can be used as a key indicator of early prediction of TPD.3. The complete open reading frame (ORF) sequence of the rubber tree glutaredoxin gene (HbGRX) is 324 bp long and encodes a protein consisting of 107 amino acids.Phylogenic analysis showed that the rubber tree HbGRX had a closest relationship with the cassava (Manihot esculenta) MeGRX and its amino acid sequence had a high homology with that of the cassava MeGRX, Ricinus communis PcGRX, Jatropha curcas JcGRXand Populus euphratica PeGRX, with a typical CPYC binding site for the protein domain of thioredoxin family (therefore belonging to CPYC type glutaredoxin). The protein was predicted to be located in the mitochondrial membrane. Protein subcellular localization study with the transient expression of the vector pCAMBIA1302- HbGRX in tobacco leaf showed that HbGRX was located in the mitochondria of the lower epidermal cells of tobacco leaf, which was consistent with the prediction.HbGRX gene expression analysis was conducted with different tissues of RY 7-33-97(an elite rubber tree clone), barks of healthy plants and TPD plants of a variety of clones,bark and latex of RY 7-33-97 plants with different severity of TPD, and with treatments of damage and different plant hormone. The tissue-specific expression showed that the highest expression level of HbGRX was found in male flowers and the lowest in mature leaves. The expression pattern of HbGRX varied with different clones of healthy plants and TPD plants.There was significant difference in HbGRX expressions in the barks of healthy plants and TPD plants of RY 7-33-97 clone, but no difference for PR107 clone. Compared with healthy trees, the expressions of HbGRX gene in the bark and latex of rubber trees with different severity of TPD decreased significantly. The expression of HbGRX gene in the bark gradually decreased with the increase of severity of TPD, while the expression in the latex decreased first and then increased. The HbGRX expression decreased after the treatment of damage, MeJA and ET, but increased first and then decreased after treatments with ABA,SA, 2, 4-D, GA3, 6-BA and IAA. After H2O2 treatment, there was no significant difference in the expression of HbGRX gene within the duration of 0-12 h, and afterwards the HbGRX expression decreased and increased significantly. These results laid a foundation for the further study on the function of reactive oxygen species (ROS) in the occurrence of TPD and on the mechanism of TPD.