Cloning And Expression Analysis Of Floral Meristem Identity Gene In Chinese Jujube (Zizyphus Jujube. Mill)

Chinese jujube or Chinese date (Zizyphus jujube. Mill) is originated from China, and is an economically important woody plant. Its fruit can be used both as food and drug. Compared with other woody fruit trees, jujube tree has a relative short juvenile phase; grafted seedlings begin to flower and set fruit on the same year of growth, seedlings from seeds flower two or three years later. Furthermore, fruit bearing shoots (FBS) are soft, delicious, fascinated on mother fruit bearing shoots; and its flower development concurs the growth of FBSs, and the whole process from flower development to fruit ripening is complete within a current growing season in the species. Cloning of floral meristem identity gene FLO/LFY, SQUA/AP1, and their expression analysis will be useful for understanding the molecular mechanism of floral formation, the control mechanism of vegetative growth and flower development in Chinese jujube.Early in 1960s, Qu et al had used "Pozao" as plant materials and observed the morphology of flower development, finding that the floral development concurs the growth of fruit bearing shoots. Nearly at the same time, Li had reported the structure and developmental character of buds and suggested that delicious FBSs developed from accessory buds in jujube tree. These studies are very helpful to further study on floral development in the species. However, the relationship among the differentiation of FBSs, the growth of FBSs, and floral development is still unknown especially in early FBSs. In this paper, the relationship between vegetative growth and reproductive development was first investigated by morphological observation using "Hupingzao" as plant material. Based on flower developmental character, we had divided FBSs into two groups: early growing FBSs and shoot apices. Secondly, a cDNA library for early growing FBSs was constructed to understand gene expression profiles and select or clone genes of interest. Thirdly, five potential housekeeping genes were screened for further expression analysis to obtain suitable housekeeping gene involved in the development of FBS and flower. Finally, homologues of floral meristem identity gene FLO/LFY, SQUA/AP1 were successfully cloned and their expression patterns were analyzed by semi-quantitive RT-PCR.Part 1: Morphological character of differentiation, growth of FBSs and flower development. The developmental morphology of FBS and flower were first observed. The results showed that FBSs developed from initiated mother bearing shoots, flower development began after FBSs sprouted, the development of leaf primordia concured floral formation in early growing FBSs, and the development of leaf primordia was complete in early growing FBSs, but shoot apices from their emergence to senescence was at developmental state of leaf primordia and floral buds. The results also showed that the structure of shoot apices was not always three germ layers, but the shoot apices possess three germ layers after flowering. The growth pattern of FBSs was obtained by measuring the length of FBSs, showing that the growth rate of FBSs is slow until basal floral buds appear, then FBSs grow rapidly, the growth of FBSs slows down and gradually cease by shedding of shoot apices after flowering.Part 2: Construction and analysis of a cDNA library for early growing FBSs. As described above, the development of leaf primordia and floral buds was not complete, and vegetative growth concurs the reproductive development at this stage. In order to acknowledge gene expression profiles and select genes of interest, a cDNA library for early growing FBSs was constructed using Superscript^TM Plasmid System with Gateway(?) Technology. The results showed that the library capacity was 1.23×10⁵, the rate of recombination was 39.9%, the cDNA inserts between 1,000～2,000 bp in length were about 31.15%, inserts between 500～750 bp were about 28.07%, and the insert larger than 2,000 bp was the least, only about 3.69%. Among 407 ESTs sequenced, 367 unisequences, and 202 uniseqs, being consisted of 248 ESTs, exhibited significant homology to genes in the nucleotide acid database at the protein level, share 67.57 percent of the whole uniseqs. 77 ESTs, about 20.98 percent had significant homology to unknown proteins or hypothetical proteins or function unknown. And 42 ESTs had no homologues in database. The iniseqs displaying significant homology to known genes were further grouped according Gene Oncology system. The expressed genes participate such biological process as metabolism, transport, cell growth, development, signal transduction, stress response, and so on. Among them, genes involved in metabolism were the most, about 36.5%, genes involved in signal transduction and stress response were the next, about 28.31% respectively. Expression profile of molecular function demonstrated that gene products function as catalytic activity, regulation of enzyme activity, signal transduction, binding, structure component, translational regulation, transport, motor, etc, and the genes involved in catalytic and binding activity are highly expressed. More than one clone of chlorophyll a/b binding protein, auxin binding protein and GTP binding protein were obtained.Part 3: Selection of housekeeping gene for gene expression study involved in the development and growth of FBSs, flower development in Chinese jujube. Four potential housekeeping genes were selected from sequenced ESTs, an actin fragment was isolated using cDNA library as PCR template. Among them, protein products encoded by ZjEF1 (accession number: EU916202), ZjCyP (accession number: EU916203), and ZjUBQ (accession number: EU916200) are involved in protein translation (translation elongation factor 1α, EF1α), protein folding (cyclophilin, CyP) or degradation (ubiquitin extension protein, UBQ) respectively. The other two, ZjAT1 (accession number: EU251881) and ZjH3 (accession number: EU916201) are essential to the structure of the cytoskeleton (β-actin, ACT) or of the nucleosome (histone3, His3). These genes are commonly used as reference genes because they exist in all nucleated cell types and they are essential to the cell survival. Using the same cDNA sample as template for different experiment, expression of ZjEF1, ZjCyP, ZjUBQ, ZjAT1, and ZjUBQ were determined in early growing shoots, shoot apices and different organs by semi-quantitative RT-PCR. The results showed that ZjH3 was the most stable internal control gene, and the other four were not suitable as housekeeping genes and their expression were related to tissue types and developmental stage in the species. The results also suggested that it is necessary to select suitable housekeeping gene for emerging organisms.Part 4: Cloning and expression analysis of floral meristem identity gene FLO/LFY in Chinese jujube. FLO/LFY is a key regulator of floral development, playing an important role in changing the differentiation fate of shoot apical meristems after flowering induction. Besides, FLO/LFY can activate floral organ identity gene. Based on conserved nucleotide acid sequence of FLO/LFY homologues, a pair primer was designed and used to isolate FLO/LFY fragment from newly expanded leaves by reverse transcript PCR (RT-PCR). A 446 bp cDNA clone, designed as ZjLFY was obtained and shared high similarity with FLO/LFY homologues both at nucleotide acid and amino acid levels. Phylogenetic analysis of FLO/LFY homologues from different core endicotyledon species showed that ZjLFY was at an independent secondary evolution point, and should be evolved in earlier time. The expression pattern demonstrated that ZjLFY was constitutively expressed in early growing shoots, shoot apices, and different tissues, showing a more extensive role than promoting flower development. The results also suggested that ZjLFY was a key regulator related to both vegetativegrowth and reproductive development.Part 5: Cloning and expression analysis of floral meristem identity gene SQUA/AP1 in Chinese jujube. SQUA/AP1 functions not only as a floral meristem identity gene, but as a floral organ identity gene specifying the development of sepal and petal. A full-length cDNA of SQUA/AP1, designed as ZjAP1 (accession number: EU916199) was obtained from sepal tissue by the methods of RT-PCR and RACE (rapid-amplification of cDNA ends). The ZjAP1 open reading frame (ORF) was 738 bp in length, including heterogeneous 3'- and 5'-untranslated regions (UTR). The deduced amino acid sequence contained 245 amino acids with typical SQUA/AP1 MADS-box conserved domains, and shared the highest identities with SQUA/AP1 homologues from Rosaceae. Expression analysis of ZjAP1 demonstrated that the expression of ZjAP1 was not constitutive and can be detected in early growing FBSs, shoot apices, axillary and floral buds, mature flowers, young fruits, roots, stems, and leaves, suggesting that ZjAP1 play a role in both vegetative growth and reproductive development in the species. The result also shows that the expression profile of ZjAP1 was narrower than that of ZjLFY.