Functional Investigation Of The HD-Zip Transcription Factor Gene Mfhb-1 In Aalfalfa (Medicago Falcata L.)

Previous research have indicated that the gene named Mfhb-1 in this study which was isolated during the induction of the early stages of somatic embryogenesis in alfalfa by subtractive cDNA cloning technology belongs to a kind of HD-zip I transcription factor gene. The similar genes have been found in other systems and associated to somatic embryogenesis. Sense and antisense technology were used to investigate the biological function of the Mfhb-1 during somatic embryogenesis in alfalfa. The transformed plants derived from 47/1-5 were generated and they are F4 (containing Mfhb-1 coding sequence), D3 (containing Mfhb-1 coding sequence in antisense orientation), C5 (containing full length Mfhb-1 sequence, including CDS and uORF).Somatic embryogenesis was inducted and record in 4 kinds of alfalfa in study. 8 kinds of hormones and 10 kinds of metal elements were surveyed by HPLC technology and ICP-AES technology; In reasearching for the expressing of Mfhb-1 gene in other model plant, the preliminary research of transforming Arabidopsis thaliana was done; The bioinformatics sciences analysis of Mfhb-1 gene was done. The main results as follows:(1) In the early development of somatic embryogenesis of alfalfa (5th day), some protuberant structure appears on the surface of F4; 47/1-5 is 10th day; D3 and C5 is 15th day. At the end of culture, somatic embryos quantity of 4 kinds plant material from low to high is D3,C5,47/1-5,F4.(2) In the 5th day of inducting culture, the lowest content of IAA of 4 plant material is F4; In the 18th day of inducting culture, the content of IAA from low to high is F4, C5, 47/1-5, D3, That the overexpress of Mfhb-1 gene maybe inhibits the increase of the content of IAA at the early of somatic embryogenesis could be inferred. But along with the culture the content of IAA is contrary with the expression of Mfhb-1 gene, this maybe could explain that Mfhb-1 gene participate in the feedback regulation of the synthesise of the IAA.(3) In the 5th day of inducting culture, the content of GA3 from low to high is F4,C5,D3,47/1-5; and keeping at a low level (1.92μg/g 2.25μg/g) in the other period of culture time. This can explain that the overexpress of Mfhb-1 inhibits the increase of the content of GA3 at the early of somatic embryogenesis, high content of GA3 is not good for the increase and grown of the embryogenesis cell. (4) In the 5th day of inducting culture, the content of ZT from low to high is C5,F4,47/1-5,D3; In the 18th day, the content of ZT from low to high is F4,C5,47/1-5,D3, This can explain that the overexpress of Mfhb-1 maybe inhibits the increase of the content of ZT at the early of somatic embryogenesis, high content of ZT is good for the increase and grown of the embryogenesis cell possibly.(5) In the 10th day of inducting culture, the content of ABA of 4 kinds of plant material from low to high is C5,D3,47/1-5,F4;In the 18th day, the content of ABA from low to high is 47/1-5,F4,C5,D3. This can explain that the overexpress of Mfhb-1 promotes the increase of the content of ABA at the early of somatic embryogenesis.(6) In the 5th and 18th day of inducting culture, the content of Ca element of 4 plant material from low to high is F4,47/1-5,C5,D3. This can explain that the overexpress of Mfhb-1 promotes the increase of the content of Ca elements of the plant material.(7) In the late of inducting culture the content of Na element of 3 plant material dropping expect D3 material, This can explain that the overexpress of Mfhb-1 gene maybe promotes the increase of the content of Na elements of the material in the somatic embryogenesis of alfalfa.(8) In the period of inducting culture, the content of Mg element of 4 plant material appears a rising trend, the peak of 4 kinds of plant material appears in D3, 10th day; F4, 15th day; C5, 15th day; 47/1-5, 18th day. This can explain that the express of Mfhb-1 maybe inhibits the increase of the content of Mg elements of the plant material.(9) In the period of inducting culture, the peak of Zn element of 4 kinds of plant material from low to high is F4,47/1-5,C5,D3. This can explain that the express of Mfhb-1 inhibits the increase of the content of Mg and Zn elements of the plant material, high content of Zn maybe is not good for the increase and grown of the embryogenesis cell.(10) Bioinformatics analysis shows that the protein coded by Mfhb-1 is an unstable hydrophilic protein without transmembrane area, is mainly distributed in the nuclear and the cytoplasmic, 4.3% lies in the mitochondrial, corresponding to the increase of mitochondrial in somatic embryogenesis of alfalfa, and has a close relationship with HDZ-M48 gene of Lotus japonicus.Based on the results presented above, it can be demonstrated that the Mfhb-1 affects the content of the hormones and metal elements of embryo: The expression and biological function of Mfhb-1 is regulated by the content of Ca element; Possible Mfhb-1 promotes the somatic embryogenesis of alfalfa by inhibiting the sythesis of IAA and GA₃ though regulating the absorption of Na element; The content of Mg and ZT is contrast with the expression of Mfhb-1, shows that the Mg and ZT may be play a negative feedback regulation in the biological function of Mfhb-1 protein.