Studies On Key Amino Acid Sites Of PPCDC Activity Of HAL3 Protein Family

HAL3 proteins are a kind of multi-functional proteins that widely exist in the eukaryotes.HAL3 proteins in Arabidopsis,tobacco,rice,corn and other plants all have 4'-Phosphopantothenoylcysteine decarboxylase?PPCDC?activity,and all the proteins are related to plant salt tolerance.PPCDC is essential for the synthesis of coenzyme A?Co A?.We predicted that HAL3 proteins in eukaryotes regulate the quantity of Co A by their PPCDC activity,and affect the downstream metabolic pathways related to Co A,thereby affect the salt tolerance of the organism.To verify our inference,we made the specific point mutations of amino acids in rice Os HAL3 protein.The complimentary tests using the E.coli?dfp temperature-sensitive mutant strain were made to determine whether the mutant amino acids affect the PPCDC activity of the protein.Through the experiments mentioned above,we screened out the amino acids which have a key impact on the activity of PPCDC in Os HAL3.To investigate the effects of the mutant amino acids in rice,the vectors with the Os HAL3 or mutant Os HAL3??Os HAL3?were constructed and transferred into the rice.Transgenic Oryza sativa plants were produced to overexpress Os HAL3 protein or different?Os HAL3 proteins.We detected the content of Co A in wild-type rice,transgenic rice overexpressing Os HAL3 protein and transgenic rice overexpressing?Os HAL3 protein,to prove the impact of some specific amino acids of HAL3 protein on its PPCDC activity in rice.The results in rice were consistent with that of complimentary tests in E.coli.Our ultimate goal is to modify the HAL3protein through amino acid mutation so as to regulate the content of Co A in rice and increase the tolerance of rice to salt stress.In the early stage of the experiment,we found that the Cys¹⁷⁶ mutation of the Os HAL3 protein caused its PPCDC activity loss in the temperature sensitivity test.Mutation of the Ala¹⁰⁸ increased the Os HAL3's PPCDC activity in temperature sensitivity test.Therefore,the plant expressing vectors carrying the Os HAL3with the Ala108 mutation or Cys¹⁷⁶ mutation were performed respectively.We obtained three kind of transgenic rice:Os HAL3??Os HAL3-108 and?Os HAL3-176 respectively.The transgenic rice“Os HAL3”overexpressing normal Os HAL3 protein was used as the positive control and wild-type rice was the negative control.We detected the amount of Co A in the three transgenic rice—Os HAL3??Os HAL3-108 and?Os HAL3-176.The results have shown that the quantity of Co A in the Os HAL3 and?Os HAL3-108 was higher than that of the wild-type rice by 40%and 67%respectively.However,the amount of Co A in the?Os HAL3-176 was26%lower than that in the wild-type rice.The quantity of free proline in the transgenic rice Os HAL3 and?Os HAL3-108 was 2.27 times and 4.41 times that of the wild-type rice.The amount of free proline in the?Os HAL3-176 was 0.65 times that of the wild-type.A series of vectors were constructed with point mutations of the rice Os HAL3,Arabidopsis At HAL3,corn Zm HAL3,and Ap HAL3 of Brevibacterium sp.Further complementary tests in E.coli temperature-sensitive mutant strains indicated that:single point mutations of Leu⁶⁶,Ala¹⁰⁸,and Met¹⁸⁴ in Os HAL3 protein increased PPCDC activity;there was no significant influence on PPCDC activity when the single point mutations occurred on Leu⁶⁶,Ala¹⁰⁸ and Met¹⁸⁴ in Zm HAL3 protein;or Glu⁶⁶,Ala¹⁰⁸ and Lys¹⁸⁴ in Ap HAL3 protein;or Pro⁶⁶,Ala¹⁸⁴ in At HAL3 protein.The mutation Asn¹⁰⁸ of At HAL3 protein has depressed the activity of PPCDC.Point mutations in position 66,108 and 184 on rice Os HAL3 protein increased PPCDC activity.However,mutations in the corresponding amino acids in Arabidopsis,maize,and Aureobasidium pullulans did not increase the activity of PPCDC.