| Woad(Isatis indigotica Fortune)is a biennial herbaceous plant.Leaves and roots of woad have long been used as traditional Chinese medicine,and are known as “indigowoad leaf” and“indigowoad root”,respectively.Owing to the fact that the growth of leaves can be influenced by the development of the flowers,suppression of the reproductive growth of woad plants is beneficial to raising the leaf yield.On the contrary,woad plants need to enter the reproductive stage as soon as possible to facilitate rapid reproduction of seeds in the breeding season.The B-class floral homeotic genes in MADS-box family are mainly involved in specifying the identities of petals and stamens.Because stamens are responsible for generating the pollen grains,it can be said that the B-class floral homeotic genes play a crucial role in seed-setting and fruit-bearing.Therefore,functional determination of B-class floral homeotic genes is of great reference value in cultivation of woad and other medicinal plants.In this work,the c DNA of two woad genes,IiAP3 and IiPI,was cloned from the inflorescences,and their functions were investigated subsequently.The main findings are as follows:(1)The open reading frames(ORFs)of IiAP3 and IiPI were cloned,and were 699 bp and 627 bp in length,respectively.IiAP3 and IiPI are B-class floral homeotic genes of the MADS-box family.(2)Phylogenetic analysis showed that IiAP3 and IiPI were highly homologous with Arabidopsis At AP3 and At PI,respectively.The encoding product of IiAP3 contains a PI-derived motif and an eu AP3 motif.The encoding product of IiPI contains a PI motif.(3)PSORT prediction showed that IiAP3 and IiPI were localized in the nucleus,and subcellular localization experiments and tissue-specific expression analyses were carried out subsequently.By using GFP(Green Fluorescent Protein)as a tag,the results of transient expression in protoplasts confirmed that IiAP3 and IiPI were localized in the nucleus indeed.IiAP3 and IiPI mainly expressed in flowers,and stamens and petals exhibited the highest m RNA abundances of these two genes,indicating IiAP3 and IiPI were mainly involved in morphogenesis of the second and third whorls of floral organs.(4)Binary expression vectors,including p CAMBIA 1302-IiAP3,p CAMBIA 1302-IiPI,pRI101-AN-IiAP3 and pRI 101-AN-IiPI,and binary expression vectors of domain swapping between IiAP3 and IiPI based on pRI 101-AN,were constructed in the present work.IiAP3 and IiPI were integrated into Arabidopsis genome by Agrobacterium-mediated floral-dip genetic transformation method,and overexpressing transgenic plants as well as back-complemented transgenic plants were prepared by screening on selection medium.Phenotypic observation was further conducted to determine the functions of these two genes.(5)The effect of IiAP3 and IiPI silencing at the m RNA level on plant growth and floral development of woad was analyzed by VIGS(Virus Induced Gene Silencing).Downregulation of IiAP3 resulted in a reduction in the number of stamens,and some flowers were infertile.Knockdown of IiPI resulted in a reduction in the number of sepals,petals and stamens,and sterility owing to incomplete development of stamens and petals.(6)The interaction of IiAP3 and IiPI with E-class proteins was examined using yeast two-hybrid techniques.By phenotypic observation to transgenic Arabidopsis plants,the present work confirms that IiAP3 and IiPI are the main regulatory factors controlling the formation of the second and third whorl floral organs.Ectopic expression of IiAP3 in Arabidopsis resulted in phenotypic changes in stamens and stigma.The third whorl floral organs in IiAP3 overexpressing plants changed from the standard ’four strong stamens and two weak stamens’ to ’five strong stamens and one weak stamens’,or even six stamens with the same length.The stigma and valve in these plants were chapped,and the ovules were exposed.In addition,IiAP3 overexpressing plants produced curly siliques and a large number of seeds were aborted.In IiPI transgenic plants,the sepals were converted into petaloid floral organs and flared out.Moreover,chimeric tissues of sepals and petals were produced.In different complemented transgenic plants of ap3-6 and pi-1 mutants,varying degrees of petal and stamen recovery could be found.In 35S::IiAP3 transgenic lines under the genetic background of ap3-6 homozygous mutant,the second whorl of the flowers produced partially normal petals.However,overexpression of IiAP3 in ap3-6 homozygous mutant could rescue the third whorl floral organs;In other words,stamens could be recovered.Under the genetic background of ap3-6homozygous mutants,chimeric genes of IiAP3 and IiPI could not completely complement the phenotypic variations;In some transgenic lines,the number of stamens was fully replenished,but these stamens only produced fewer pollen grains;In other transgenic lines,only two stamens were recovered;In like manner,petals could only be partially rescued,the number of petals varied between 2-4,and the relatively narrow petals were different from wild-type petals in shape;In addition,partial transgenic plants produced misplaced petals and fused stamens.In 35S::IiPI transgenic lines under the genetic background of pi-1 homozygous mutant,normal petals were produced in the second whorl of the flowers,but the third whorl floral organs were not completely rescued,and the development of stamens was abnormal.In general,stamens are composed of filament and anther.In 35S::IiPI transgenic lines under the genetic background of pi-1 homozygous mutant,only the filaments were recovered.Because no pollen grains could be produced in these stamens,most flowers were sterile.Moreover,chimeric tissues of stamen and pistil could be observed in a portion of 35S::IiPI transgenic plants under the genetic background of pi-1 homozygous mutant.In these transgenic plants,filaments clung tightly to the carpel,and saclike green anthers were formed.In summary,the present work focused on cloning of IiAP3 and IiPI,two B-class floral homeotic genes in woad,and on verification of their functions in reproductive stage.The obtained results possess important theoretical implications for guiding the improvement of the seed reproductive capacity by promotion of flowering,and the increase of leaf yield by delaying the flowering time. |
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