【作者】 安颖慧；

【导师】 李小方；

【作者基本信息】 华东师范大学 ， 植物学， 2007， 硕士

【摘要】 CAL(CAULIFLOWER)基因与AP1(APETALA1)基因都是控制花分生组织特性的基因，二者都属于MADS-box转录因子编码基因。在拟南芥中，CAL基因单突变时没有明显的表型，但AP1，CAL同时突变会使花分生组织保持花序分生组织的无限分生特性，大量增生分生组织结构，形成花球。而花椰菜(Brasscia oleracea L.var.botrytis)中BobCAL基因单突变就能形成花球，显然两个物种中CAL和AP1同源蛋白的功能有所不同。即使作为花分生组织特性的控制基因，人们对CAL和AP1的靶基因也不清楚。为了研究芸苔属植物中CAL和AP1同源蛋白的功能，尤其是在调控顶端分生组织方面的功能，我们从分析它们的互作因子和遗传学两个方面进行了初步的探讨。一方面，采用酵母双杂交的方法从分子水平筛选与芸苔属中BoCAL互作的因子，结果得到互作较强的四类蛋白，分别涉及蛋白质的磷酸化和去磷酸化、蛋白质的修饰、蛋白质的结合位点等，它们分别与转录调控途径及信号转导途径有着密切的联系，这些因子的获得为BoCAL作用机制的研究提供了线索。选取筛选到的与BoCAL互作最强的蛋白，检测它与BobCAL不同剪切之间的互作关系，结果没有互作现象，说明BoCAL与BobCAL的互作因子存在不同，说明BobCAL部分K-box及其后的C-端的缺失影响了蛋白质的互作，为了探究该突变基因是否还有花分生组织特性决定性，我们构建了BoCAL和BobCAL两个基因的过量表达载体，并通过转基因方法获得得这两个基因的拟南芥中过量表达植株，结果发现BoCAL过量表达时，可以显著促进拟南芥提前开花，而BobCAL过量表达时，也能一定程度上促进开花提前，这表明和全长的BoCAL相比，花椰菜中BobCAL发生的基因提前终止，导致其在促进开花方面的功能有所削弱，但是并没有完全丧失，这种花分生组织特性决定性的功能削弱可能是花椰菜花序发育过程需要长时间积累BobCAL从而导致发育停滞、花球形成的原因。为了分析CAL和AP1同源蛋白在芸薹属和拟南芥中的功能差异，我们同样利用酵母双杂交方法检测了部分MADS-box转录因子和BoCAL,BoAP1之间的互作关系，结果表明，BoCAL,BoAP1和拟南芥中同源蛋白都能与SVP互作，但与拟南芥中同源蛋白不同的是，BoCAL,BoAP1与FLM,SOC1(SUPPRESSOR OF CO OVEREXPRESSION 1)和AGL24(AGAMOUS-LIKE24)作用很弱或不能互作，暗示BoCAL和BoAP1与拟南芥中同源蛋白功能上是不完全相同的。在SVP的克隆过程中，克隆到了SVP的一个新的剪切SVPa，它与BoAP1不能互作，可能与SVP有不同的功能。对SVP和SVPa的荧光定位发现它们都在细胞核及细胞膜中表达，结合它们的可变剪切的多样性和表达时间的持久性，说明其表达受到了精密的调控，暗示SVP在植物发育过程中功能相当重要。荧光共定位的方法对AP1和SVP在植物体内互作关系进行了进一步验证。同时利用分子生物学方法对这两个基因在表达水平的相互调节关系进行了初步分析。结果发现，AP1过量表达植株的花中，SVP的表达量下降；svp突变体中，AP1的表达量也会出现下调，表明，AP1对开花的影响可能主要通过SVP来进行调节，而SVP对开花的影响可能受到了其他的调控。另一方面，为了研究CAL、AP1和控制顶端分生组织的相关基因间的遗传关系，我们通过遗传杂交的方法将ap1，ap1cal突变体与控制顶端分生组织的WUS-CLV途径中重要相关基因的突变体进行了杂交，ap1wus双突变表现wus单突变的表型，顶端分生组织的发育受到抑制，说明ap1突变体顶端分生组织的增生是WUS依赖性的。当它们与clv进行杂交之后，表型增加，或者出现两种突变体表型叠加的现象，说明他们是在两条不同的途径中起作用。而分子生物学RT的结果表明，ap1突变体中WUS的表达量没有明显变化，而ap1cal突变体中，WUS的表达量出现下调，暗示着CAL的参与对WUS的表达情况有了更为复杂的调控。以上对CAL、AP1同源蛋白功能的初步分析进一步提供了花椰菜花球形成基础的分子证据，同时互作因子的获得为进一步探讨花球形成指明了方向，本工作第一次对花球的形成与控制顶端分生组织相关基因间的遗传关系进行了初步分析，这部分工作的深入研究将为CAL、AP1基因在调控顶端分生组织方面的功能提供突破口。更多还原

【Abstract】 CAL (CAULIFLOWER) and AP1 (APETALA1) g, both of which belong to MADS-box gene family, are floral meristem identity gene. In Arabidopsis thaliana, loss of cal function doesn’t result in obvious phynotype, while loss of cal, ap1 function at the same time leads to floral meristem proliferation unlimited, and displays the cauliflower phenotype. But as to cauliflower (Brasscia oleracea L.var.botrytis) , the BobCAL single mutant can display the cauliflower phenotype. It appears that CAL and AP1 homolog have different function in both species. Even as regulator of the flower apical meristem, we know few about of CAL and API’s target genes. To study the function of homozygous CAL , AP1 protein in Brassica Species, especially their regulater function in the apical meristem, we probed into this question from molecular and genetic two sides.On one hand, yeast-two-hybrid method was applied to screen interaction factors of BoCAL. We got four kinds of proteins which are respectively involve in phosphorylation and dephosphorylation of proteins, proteins modification, proteins binding-site and so on. They separately have close relationship with translation, regulation pathways and signal transferring pathways respectively, which provide clues for the study of function of BoCAL. We choose the most strong interaction factor of BoCAL, and testify the relationship between the factor and different splicing of BobCAL, the results showed there is no interaction relationship between them. This suggests that there are different between BoCAL and BobCAL’s interaction factors, and then the absence of partial K-box ang C terminance. To study if BobCAL with mutation still has function in floral meristem determinance, we construct overexpression vectors of both BoCAL and BobCAL,and obtained overexpressed transgenic plants in Arabidopsis. The results showed that overexpressing BoCAL can greatly promote flowering, while overexpressing BobCAL also promoted flowering to some extent. This indicates that compared with the full lengh BoCAL, the function of BobCAL which terminated prematurely in cauliflower was weakened, but not lost in the aspect of promoting flower. The weakened function of BobCAL may be the reason of cauliflower’s inflorescence stop development for a long time and arrising a curd, and then it can accumulate enough BobCAL to promote flowering.Additionally, to analyze the difference of the homolog of CAL and AP1’s function in Brassica and Arabidopsis, we testify the relationship between some known MADS-box translation factors and BoCAL and BoAP1. The results showed that both BoCAL and BoAP1 interact with SVP, while there were no interaction between BoCAL, BoAP1 with FLM, SOC1 and AGL24. The data here indicate the function of homozygous BoCAL and BoAP1 differ from that in Arabidopsis thaliana.During the cloning of SVP, we abtained a new splicing of SVP, it can’t interact with BoAP1, and then it may have different function with SVP. We found both of them express in cell membrane and nuclear using the fluorescence microscopy method. Considering SVP has kinds of splicing and it express during almost all stage of the plant, there must be exact regulation during the expression of SVP, and it indicates SVP has important function during the plant development.Using the fluorescence microscopy method,we further validate the interaction of SVP and AP1 in plant. At the same time, we analyzed the interregulation of AP1 and SVP with molecular biology method. We discovered in AP1 overexpression plant, SVP’s expression is down, while in svp, AP1’s expression is down too. It suggests that AP1 influences flowering may depend on SVP, but SVP influences flowering through other pathway.On the other hand, we studied the genetic relationship between CAL, AP1 and genes relevant regulating apical meristem. We crossed ap1, apical mutants with mutants which are important in WUS-CLV pathway. The progeny of ap1wus displayed the phenotype of wus single mutant, the development of apical meristem is repressed, which suggested that the proliferation of ap1 apical meristem is WUS dependent. But when ap1, apical were crossed with clv mutant, the clv mutant phenotype was enhanced, and displayed the character of both ap1 and clv3,which indicate these genes may function in different pathways to regulate the size of apical mesitem. The RT results showed WUS expression in ap1 has no obvious different with wild,while in apical, the expression level is down regulate. It suggests there is more complicated regulation while CAL mutates.The analysis to the homolog of both CAL and AP1 provide molecular proof to the curds arrising of cauliflower, and the analysis to the interfactor of them point out a way to the curds arrising too. Our work analyzed the genetic relationship between the curds arrising and apical meristem regulater. The further study of it will provide direction to the function of AP1, CAL to the apical meristem.更多还原