【作者】 何男男；

【导师】 周岩；

【作者基本信息】 河南科技学院 ， 作物遗传育种， 2010， 硕士

【摘要】 本课题所研究的Mfhb-1基因是在苜蓿体细胞胚胎发生早期经扣除杂交得到的,前期研究表明该基因属于同源异型域-亮氨酸拉链蛋白（homeodomain-leucine zipper protein, HD-Zip）家族I类转录因子基因。与其相似的HD-Zip基因已在多种物种中发现并与体细胞胚胎发生有关。为了研究Mfhb-1转录因子的生物学功能,在前期工作中,以苜蓿品系47/1-5为起始材料,应用正义、反义转化技术得到了转化植株。它们分别为:D3,携带Mfhb-1基因编码区反向序列的反义转化植株;F4,携带Mfhb- 1基因编码区序列的正义转化植株;以及携带Mfhb-1基因全长序列的正义转化植株C5。本文对上述3种转化材料D3、F4、C5和未转化的原始材料47/1-5进行了体细胞胚胎发生诱导,并对其体细胞胚胎发生过程进行了观察;利用HPLC技术和ICP-AES技术对苜蓿体细胞胚胎发生过程中的8种内源激素和10种金属元素的含量进行了测定;为了探索Mfhb-1基因在苜蓿以外的其他模式植物中的表达方式,对Mfhb-1基因转化拟南芥进行了初步研究;并对Mfhb-1基因进行了生物信息学分析。其主要研究结果及结论如下:（1）在苜蓿体细胞胚胎诱导发育早期,F4、47/1-5、D3和C5表面的突起结构形成时期分别为诱导培养的第5 d、10 d、15 d、15 d。在发育培养结束后（发育培养30 d） 4种植物材料所产生的体细胞胚胎数量由低到高分别为:D3、C5、47/1-5、F4。（2）在体细胞胚胎发生诱导培养的第5 d时4种材料中F4的IAA含量最低;在诱导培养的第18 d时4种材料体内IAA含量由低到高分别为D3、C5、47/1-5、F4。可以推测出Mfhb-1基因的过量表达在苜蓿体细胞胚胎发生初期可能抑制了IAA的合成,而随着培养时间的延长,培养物的生长,Mfhb-1基因表达情况又与IAA含量成反比关系,由此可推测出Mfhb-1基因的可能参与了IAA合成的反馈调节。（3）在诱导培养的第5 d时4种材料的GA3含量由低到高分别为F4、C5、D3、47/1-5,并在随后的整个培养过程保持在较低水平（1.92μg/g 2.25μg/g）。可以说明Mfhb-1基因的过量表达可能在苜蓿体细胞胚胎发生过程中抑制了GA3的合成。（4）在诱导培养的第5 d时,4种材料的ZT含量由低到高分别为C5、F4、47/1-5、D3;在诱导培养的第18 d时4种材料的ZT含量由低到高分别为F4、C5、47/1-5、D3。可以推测Mfhb-1基因的表达在苜蓿体细胞胚胎发生初期抑制了ZT的合成。（5）在诱导培养的第10 d时,4种植物材料的ABA含量由低到高分别为C5、D3、47/1-5、F4,在诱导培养结束（18 d）时,4种植物材料的ABA含量由低到高分别为47/1-5、F4、C5、D3,结合体细胞胚胎发生情况可以说明Mfhb-1基因的表达在苜蓿体细胞胚胎发生初期可能促进了ABA的合成。（6）在诱导培养的第5 d和第18 d,4种材料中Ca元素的含量由低到高均表现为F4、47/1-5、C5、D3,这可以说明Ca元素对胚性细胞的形成可能有一定的促进作用,也可推测Mfhb-1基因的表达很可能促进植物材料对Ca元素的吸收。（7）在诱导培养的后期除D3外其余3种材料的Na元素含量均表现出下降的趋势,可以推测出Mfhb-1基因的表达可能在苜蓿体细胞胚胎发生诱导的初期促进了植物材料对Na元素的吸收。（8）Mg元素含量在苜蓿体细胞胚胎诱导培养时期主要表现为上升的过程,D3的峰出现在第10 d,C5和F4的峰出现在第15 d,47/1-5出现最晚,在诱导培养的第18 d,可以推测Mfhb-1基因的表达在体细胞胚胎诱导初期可能抑制了Mg元素的吸收。（9）4种材料在诱导培养期间的Zn元素含量峰值由低到高分别为F4、47/1-5、C5、D3,说明Mfhb-1基因的表达在苜蓿体细胞胚胎诱导初期可能抑制了植物材料对Zn元素的吸收。（10）生物信息学分析表明Mfhb-1基因编码的蛋白属于不稳定亲水性蛋白,无跨膜区,主要分布在细胞核和细胞质中,还有少量在线粒体上,与苜蓿体细胞胚胎发生过程中首先观察到的线粒体增殖现象相对应,说明Mfhb-1基因可能是通过促进苜蓿胚性细胞内线粒体的增殖来促进苜蓿体细胞胚胎发育进程的;与百脉根（Lotus japonicus）的HDZ-M48基因同源性达（90.82%）,在系统发育上亲缘关系较为接近。以上结果表明,Mfhb-1转录因子基因的表达对苜蓿体细胞胚胎发生过程中的内源激素的合成和金属元素的吸收产生影响。可以推测Mfhb-1基因的生物学功能的发挥可能受到Ca元素的调节;Mfhb-1基因可能通过调节苜蓿对Na元素的吸收来抑制IAA、GA₃的合成来达到促进苜蓿体细胞胚胎的发生;Mg元素和ZT的含量与Mfhb-1基因的表达量相反,说明Mg和ZT可能对Mfhb-1蛋白生物功能的发挥有负反馈调节的作用。更多还原

【Abstract】 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.更多还原