Mechanism Analysis Of Resistances To Aluminum And Salt In Ced-9/Bcl-2 Transgenic Tobacco And Rice

Ced-9 is a Bcl-2 analog from Caenorhabdities elegans that promotes cell survival.It has been shown that apoptotic suppressors such as Bcl-X_L and Ced-9 enhance the resistance of plant cells to biotic or abiotic stresses,but detailed mechanisms still are largely unknown.Here,we choose tobacco(Nicotiana tabacum L.)as an excellent model for studying various aspects of Al or salt induced programmed cell death(PCD).And we want to uncover the mechanism and enhance the resistant to aluminum (Al)and salt stress by overexpressing Ced-9/Bcl-2 in tobacco and rice. Results obtaind in the present study are summarized as follows:1.We proved a process of PCD induced by Al in tobacco by DNA ladder analysis.2.Data of root growth length,hematoxylin staining in treatment solution containing different concentrations of Al indicate that tobacco cells overexpression Ced-9 gene are more resistance to Al.Detailed analyses show that Ced-9 reduces Al uptake amounts in plant cells.3.Afterwards,total DNA were extracted from plant cells treated with Al or salt,and DNA ladder analyses suggest that Ced-9 increases tolerance via blocking Al-or salt-induced PCD.4.Caspases as a character of apoptosis,plays an indispensible role in animal cells.Here,we show Ced-9blocks Al-or salt-induced programmed cell death(PCD)by downregulation of Al-or salt-induced transcriptional levels of Caspase-like vacuolar processing enzyme(VPE).Our research suggest that negative regulation of PCD probably is one of the potential Al-resistance mechanisms and has a broad-spectrum resistance to abiotic stresses in plant cells.5.Up to now,little is clearly understood about Al-resistance mechanism involving intracellular signal transduction and regulation mechanisms.Staining of Al-treated roots with 2',7' -dichloro fluorescein diacetate(H₂DCFDA)had suggested that Al treatment causes ROS production.Ced-9 may indirectly advance the repression of the lipid peroxidation caused by Al stress in the early process of Al-elicited ROS. Our data suggest that Ced-g acts downstream of oxidative burst to Al stress and delays the elevation of[Ca²⁺]_cytcaused by Al stress and Ced-9 enhances Al tolerance probably via delayed elevation of intracellular Ca²⁺levels and inhibition of Al-induced PCD in plant cells.6.NtGDI1,parB,NtPox are Al-induced early response genes. Overexpression of these genes could diminish the oxidative damage caused by Al stress.In our study,Ced-9 may indirectly advance the repression of the lipid peroxidation caused by Al stress in the early process of Al-elicited reactive oxygen species(ROS).7.In this paper,we also transferred Ced-9 and Bcl-2 genes into rice and by Agrobacterium tumefaciens EHA105.The results are showed as followes:(1)Using recombination techniques,the gene fragment Bcl-2 was incorporated into pGEM-7zf(+)and formed a new vector pGEM-Bcl2,in order to get the right endonuclease sites.And then the Bcl-2 gene was inserted into plant expression vector pCAMBIA13011 under the control of CaMV 35S promoter.(2)The Ced-9 and Bcl-2 genes were transformed into rice calli by Agrobacterium tumefaciens EHA105.PCR analysis,hygromycin selection and Gus assay of transformants confirmed that the Ced-9 and Bcl-2 genes have been integrated into the chromosomal DNA of these transgenic plants.(3)T1 seeds were harvested and obtained homozygotic lines of transgenic rice after hygromycin selection.Then we confirmed transgenic lines expressing Ced-9 and Bcl-2 by RT-PCR. (4)Tail-PCR(thermal asymmetric interlaced PCR)was used to analyze the locus of T-DNA insertion.In the transgenic rice,we determined the loci of T-DNA insertions in one line.T-DNA was predicated to insert the chromosomeâ…§.