Establishment Of Regeneration System Of Peanut Embryonic Leaflet And AhZAR Family Function Study

Peanut（Arachis hypogaea L.） is one of the important oil-bearing and economic crops in China.It has the advantages of wide adaptability and high economic benefits.Its total yield ranks first among oil-bearing crops.As a strictly self-pollinated crop,peanut has a very narrow genetic base.Meanwhile,peanut genome is relatively large（-2.7G）,and tissue culture and genetic transformation are difficult and genotype-dependent.Therefore,the establishment of efficient and stable regeneration and transformation system is an important prerequisite for peanut gene function research.In this study,embryo leaflets of peanut accessionss Yuanza 9102,Fenghua 2,and Hua-814 were used as explants,and the optimal medium for inducing adventitious buds of peanut embryo leaflets was established by evaluating the difference of various hormone ratios and genotypes.At the same time,the genetic transformation conditions mediated by Agrobacterium tumefaciens,such as genotype,explant,infection and co-culture time,were screened,and the improved peanut genetic transformation system was established.In addition,bioinformatics and functional analysis of the AhZAR family,important functional genes that affects the development of peanut embryos,were carried out.The main results are as follows:（1）Establishment of the peanut regeneration systema with wide adaptability and genotype independence.The results showed that the induction effect of 6-BA and NAA was better than that of TDZ,and the optimal medium for adventitious bud induction was MS+4.5 mg/L 6-BA+0.2 mg/L NAA.The medium was tested by different genotypes,of which Fenghua 2,Yuanza 9102,and Hua814 had higher adventitious bud induction rate with 75.60%,66.17%,and 56.67%,respectively.The best elongation medium was MS+4 mg/L 6-BA,and the adventitious bud regeneration rate of the three genotypes was higher than 64%.Using the rooting medium containing 0.2 mg/L IBA+0.1 mg/L NAA,the rooting rates of Fenghua 2,Yuanza 9102,and Hua 814 were 73.80%,60.32%,and 50.66%,respectively.（2）Improvement of peanut genetic transformation system.The results showed that shaking time,co-culture time and hormone concentration all affected the transformation efficiency,and the highest transformation rate was 14.71%under shaking culture for 30 min.By co-culturing for 3 days The higher transformation rate（9.50%）and the lower pollution rate,was observed.When the medium hormone ratio was 3 mg/L 6-BA+0.25 mg/L GA,the transformation rate was 10.81%.The GUS activity rate of intact embryos was the highest（58.70%）.（3）Bioinformatics analysis and tissue expression of the AhZAR family,affecting the embryonic development of peanut.The study showed that there were 8 ZAR gene members in the whole peanut genome,all belonging to the LRRⅢ subgroup of the LRR-RLK family,among which AhZAR₄ and AhZAR₈ and the AtZAR1 gene of Arabidopsis thaliana were clustered togeather.The amino acid sequence of the peanut ZAR gene is highly similar to that of AtZAR1,both of which have the typical LRR-RLK family extracellular LRR domain,transmembrane domain and intracellular kinase domain,among which AhZAR₄ and AhZAR 8 have CaM-binding motif and Gβ-binding motif similar to A tZAR1.Expression profile showed that AhZAR genes were mainly expressed in young tissues of peanut,such as bud,stamen,tips of subterranean gynophore,and seeds at the middle stage of pod development.（4）Functional analysis of AhZAR₄ in peanut.Whole genome sequencing of wild type（H2014）and mutant（H1314）showed that C>T conversion occurred at 907 bp in the first exon of AhZAR₄ gene,leading to a premature protein with only the LRR domain.The three-dimensional structure of the protein only shows obvious LRRNT 2 domain characteristics.Subcellular localization indicated that AhZAR₄ was expressed on the cell membrane,but no fluorescence signal was detected for the AhZAR₄ mutant allele from H1314.Expression profile showed that AhZAR₄ was significantly down-regulated in the mutant at the early,middle and late stages of pod development.Furthermore,the gene was induced by hormones such as BR,IAA,SA,ABA and JA.The expression of AhZAR₄ displayed positive feedback on BR and IAA,while the negative feedback on SA,ABA and JA was observed.In addition,we also found that the homologous gene AhZAR₈ can compensate for the loss of function of AhZAR₄ to some extent in the mutant.