Generation Of AOX-overexpressing Plants And Identification Of Mutants And Preliminary Analysis Of AOX2 Function In Arabidopisis Thalinan

In addition to the cytochrome pathway（CP）,plant mitochondria have an alternative pathway（AP）.Its terminal oxidase is alternative oxidase（AOX）,which is encoded by nuclear genes.There are five different genes in the Arabidopsis AOX gene family,they are AOX1 a,AOX1b,AOX1 c,AOX1d and AOX2,respectively.At present,the research on the function of AOX gene family is mainly focused on AOX1 a in plant stress resistance.In order to further explore the role of AOX family genes in growth and development of Arabidopsis thaliana,we screened and identified the AOX family related genes deletion mutant and constructed overexpression plants of AOX1 a and AOX2 with Col-0.On this basis,we explored the role of AOX2 in the process of seed germination under the existence of exogenous ABA.The results are summarized as follows:1.T-DNA mutant insertion site analysis was conducted on the T-DNA mutant lines of the Arabidopsis thaliana AOX genes（aox1a: SALK₀84897,aox1c: CS804611,aox2: CS766955,all purchased from Arabidopisis Biological Resource Center）.DNA genotyping and expression levels were carried out in order to confirm homozygous mutant plants.2.The cDNA was amplified by reverse transcription PCR（RT-PCR）from RNA extracted from Arabidopsis thaliana wildtype（Columbia,Col-0）seedlings.With the help of high fidelity enzyme,the two target AOX genes AOX1a（1065 bp）and AOX2（1062 bp）were amplified by PCR and cloned into the p-Blunt vector and the sequences of the genes were confirmed by sequencing.The PCR products of confirmed AOX1 a and AOX2 were then cloned into the Entry Vector and sequenced.3.The expression vector pGWB2 was used to generate Arabidopsis AOX1 a and AOX2 overexpression constructs based on the Gateway technology.The constructs were confirmed by sequencing and transformed into Agrobacterium tumefaciens.4.The AOX1 a and AOX2 overexpression plants were obtained by Agrobacterium-mediated transformation.Transgenic plants were selected based on antibiotics Hyg resistance and the expression level was examined by real-time quantitative PCR.5.The results of seed germination showed that: in 2 days after germination,the germination rates of Col-0,aox2 and two AOX2 over-expressing lines OE#1 and OE#4 on MS were 94%,80%,100% and 96%,respectively;compared with the control,seed germination rates was reduced to 60%,46%,86%,and 72%,respectively,when 1.0 μM exogenous ABA was added to the media.The results showed that under ABA treatment,the mutant aox2 showed increased sensitivity to ABA,but the overexpressing plants had decreased sensitivity.6.By adding different concentrations of ABA,the seeds of Col-0,aox2 and OE#1 and OE#4 were germinated in the dark for 5 days and then the germinated seedlings were transferred to the light.Two days later,the seedlings of Col-0,aox2,OE#1 and OE#4 germinated and grown on the normal MS media were found to have a greening rate of 100%;however,the greening rate of cotyledon decreased significantly under 1.0 μM ABA treatment,with OE#1 and OE#4 decreasing to 70%,and Col-0 and aox2,to 42% and 34%,respectively.7.After germinating for one day,the germination rates of Col-0,aox2,OE#1,and OE#4 were 74%,54%,78% and 76%,respectively.By adding 1.0 μM of GA3,the germination rates of Col-0,aox2 OE#1,and OE#4 were 44%,36%,74% and 56%,respectively.When 1.0 μM ABA and 1.0 μM GA3 were added,the germination rates of Col-0,aox2,OE#1 and OE#4 were 56%,44%,72% and 66%,respectively.Comparing to the germination rates with the addition of 1.0 μM ABA alone,the germination rates under ABA and GA3 increased by 4%,2%,14% and 7%,respectively.The results indicated that ABA inhibition on seed germination is obviously relieved by adding GA3.8.The results of gene expression analysis showed that AOX knockout and overexpression had no effects on the expression of ABI5,NCED6,and RAB18;however,mutation of AOX2 up-regulated the expression of ABI3,ABI4,RD29 A,RAB18,and MYC2 in the seeds,whereas overexpression of AOX2 decreased the expression of the ABA signaling genes.