Recombination Of ABF1,ABF2,and ABF4 Genes Of ABI5 Subfamily And Construction Of Overexpressed Transgenic Arabidopsis

Twenty years have passed since the identification of the fifth Arabidopsis thaliana mutant(ABA-insensitive mutant)gene ABI5 that does not respond to ABA.As a leucine zipper type(bZIP)transcription factor,there are 8 genes that are highly homologous to ABI5 in the Arabidopsis genome,and they are collectively classified as ABI5 subfamily(ABI5 subfamily).ABI5 subfamily genes are involved in many stages of plant growth and development,such as seed maturation,seed germination,root growth,flowering time,and stress response.Among them,ABF1,ABF2,and ABF4 are the first three family members of the ABI5 subfamily.The differences in their biological functions and molecular mechanisms of their functions are still unclear.Therefore,it is necessary to systematically study the expression patterns of the ABFs bZIP genes of this subfamily member.In order to avoid gene redundancy while being able to see the contribution of a single mutant among multiple mutants to the phenotype,based on the previous work of this laboratory that proved the highest CRII activity in the transcriptional activation region of AtDPBF4,this article connects AtDPBF4 The transcription activation region CRII and the basic leucine zipper region of ABF1,ABF2,ABF4 obtain the recombinant transcription factor with transcription activation activity.By constructing plants overexpressing the recombinant transcription factor genes,we hope to understand the effects of ABF1,ABF2 and ABF4 on plant growth and adversity adaptability.Through analysis of Arabidopsis plants(A.thaliana A1)overexpressing the recombinant gene 4IIA1bZIP,the following results are obtained in this paper:1.The seed germination of Arabidopsis plants overexpressing the recombinant gene 4IIA1bZIP was significantly earlier:After 44 hours of spotting on 1/2 MS medium,the germination rate of the seeds was observed.Statistics found that the germination rates of wild-type plants at 44h,45h,46h,47h,and 48h were 10%,11%,13%,15%,and 19%,respectively;the germination rates of transgenic plants were 14%,18%,36%,respectively.43%,45%.2.The height of Arabidopsis plants overexpressing the recombinant gene 4IIA1bZIP increased:A.thaliana A1 transgenic Arabidopsis lines(A.thaliana A1-2,A.thaliana A1-6 and A.thaliana A1-10)entered the end of the growth cycle at 8 weeks.At this time,the average plant height of the transgenic line A.thaliana A1-2 was 42 cm,the average plant height of A.thaliana A1-6 was 40 cm,the average plant height of A.thaliana A1-10 was 44 cm,and the wild-type plant height was 35 cm.3.The bolting number of Arabidopsis plants overexpressing the recombinant gene 4IIA1bZIP increased:The T4 generation seeds of A.thaliana A1 transgenic Arabidopsis strains(A.thaliana A1-2,A.thaliana A1-6 and A.thaliana A1-10)were cultured in the greenhouse,and the Arabidopsis grew for 8 weeks and entered the end of the growth cycle The results showed that the bolting numbers of Arabidopsis plants overexpressing the 4IIA1bZIP gene all showed a phenotype ranging from 3 to 4.4.The drought resistance of Arabidopsis plants overexpressing the recombinant gene 4IIA1bZIP was significantly weakened:Three-week-old wild-type and overexpressing Arabidopsis(A.thaliana A1-2,A.thaliana A1-6 and A.thaliana A1-10)seedlings were treated with drought for 10 days,A.thaliana A1 transgenic Arabidopsis seedlings leaves are yellow,and the wild-type leaves appear wilting.After one week of rehydration,A.thaliana A1 transgenic Arabidopsis seedlings dried up and stopped growing,but the wild-type wilting state gradually disappeared and growth resumed.5.The salt tolerance of Arabidopsis plants overexpressing the recombinant gene 4IIA1bZIP may be enhanced:Spot the seeds on 1/2 MS medium with different NaCl concentrations and observe the condition of Arabidopsis after two weeks of growth.It is found that wild-type germination is delayed on 1/2 MS medium with 50 m M and 100 m M NaCl.The wild type on 1/2 MS almost stopped germinating,while the transgenic plants took longer to germinate but could continue to germinate.6.The number of rosette leaves in Arabidopsis plants overexpressing the recombinant gene 4IIA1bZIP increased:Observe the number of rosette leaves of wild type and Arabidopsis thaliana seedlings overexpressing the 4IIA1bZIP gene after two weeks of growth: wild type has 11 rosette leaves;transgenic line A.thaliana A1-2 has 14 rosette leaves,A.thaliana A1-There are 16 rosette leaves of 6 and 14 rosette leaves of A.thaliana A1-10.7.Arabidopsis plants overexpressing the recombinant gene 4IIA1bZIP are more sensitive to ABA:Spot the seeds evenly on 1/2 MS medium containing different concentrations(0?M,0.5,1,5,10?M)of ABA.After one week of growth,the transgenic plant A.thaliana A1 failed to germinate.After another two weeks of observation,it was found that on 1/2 MS with 0.5 ?M ABA,the number of germination days increased.Among them,the wild type grew rosette leaves,and about 80% of the seeds of the transgenic plant A thaliana A1 failed to germinate.8.Arabidopsis plants overexpressing the recombinant gene 4IIA1bZIP are more sensitive to sucrose:Sterilize the seeds on 1/2 MS containing different percentages of sucrose.Observe the growth of Arabidopsis thaliana two weeks later.It is found that the wild-type plants grown on 1/2 MS medium containing 1% and 3% sucrose showed no significant difference in cotyledon size and root length from transgenic plants.The germination time of transgenic plants on 1/2 MS containing 6% sucrose was significantly longer than that of wild type,and the root length and the rosette leaves became shorter and smaller.9.The cold resistance of Arabidopsis plants overexpressing the recombinant gene 4IIA1bZIP is significantly enhanced:Seedlings of wild-type and transgenic lines(A.thaliana A1-2,A.thaliana A1-6 and A.thaliana A1-10)grown for three weeks were treated at 4? for 7 days and then moved to a suitable growth temperature(23?)for one week,it was found that the growth of the transgenic plants improved and the wild type died.10.The Arabidopsis plants overexpressing the recombinant gene 4IIA1bZIP did not participate in osmotic stress:Sterilize the seeds on 1/2 MS medium containing different concentrations of sorbitol.Observe the wild-type and over-expressing recombinant gene 4IIA1bZIP strains that have grown for two weeks.It is found that 50 m M,100 m M,200 m M,and 400 m M sorbitol11.Among the nine members of the ABI5 subfamily,ABF1,ABF2,ABF3,and ABF4 have self-activating activity,while AtDPBF1,AtDPBF2,AtDPBF3,AtDPBF4,and At5G42910 have no self-activating activity.