Cloning And Identification Of Genes Related To Self-incompatibility Of Citrus Grandis Var. Shatinyu Hort. Styles

Being one of the important fruit trees in south China, the shaddock (Citrus grandis var. Shatinyu Hort.) tree is a typical gametophytic self-incompatibility (GSI) plant. Its lower yields has greatly limited the development of the shaddock production. The investigation of the molecular mechanism of GSI and the cloning of the key genes related to self-incompatibility (SI) of Citrus grandis var. Shatinyu Hort. are of great value in both shaddock production and the scientific research. In this study, through adopting suppression subtractive hybridization (SSH) differential screening technique (Fig. 3-3), we constructed a forward subtractive cDNA library of the self-pollinated styles in Citrus grandis var. Shatinyu Hort.. SSH was performed by using the self-pollinated style as tester and the cross-pollinated style as driver. Consequently, expressed sequence tag (EST) of genes related to SI of Citrus grandis var. Shatinyu Hort. styles were cloned. After the SSH library was further screened by use of the PCR technology combined with extracting and digesting method to recombined plasmids, partial positive clones were picked up randomly and sequenced. Then the sequences homology analysis was performed in unigene database of GenBank with Blastx and Blastn algorithm from National Centre for Biotechnology Information, showing that genes involving in SI response were found. All these studies will help us to better understand the molecular mechanism of GSI in Citrus grandis var. Shatinyu Hort..Our results of study are as follows:The experiment of the white and blue blot screening proved that the SSH library contained 247 unique clones in all (Fig. 3-4), of which the rate of recombination was higher than 95%. Further with the PCR analysis(Fig. 3-5) and the identification of extracting and digesting to recombined plasmids (Fig. 3-6), 128 positive clones were testified from forward subtractive cDNA library, and the length of the inserted fragments ranges from 100 bp to 1.0 kb. From these clones 120 positive clones were picked up randomly and sequenced. After analysing the nucleotide sequences and removing redundant cDNAs, 30 kinds of EST sequences (ESTs) were obtained. Among these ESTs, 76.7% including 23 kinds of ESTs had significant homology with known genes through being compared with sequences in unigene database of GenBank with Blastx algorithm (Table 3-1).With the analysing of the 23 ESTs, it was supposed that hect E3 ubiquitin ligase and protein kinase would play a role in the SI response. The former was likely to work by the ubiquitin and 26S proteasome pathway, while the latter was likely to work in the SI response by the cell signal recognition and conduction pathway of phosphorylation and dephosphorylation of protein. Furthermore, we found several ESTs in the forward subtractive cDNA library, which probably supplied evidence for the hypothesis of"membrane receptor"from molecular level. These ESTs are stigma expressed protein, plasma membrane H+ ATPase, rRNA intron-encoded endonuclease and membrane related protein.However, the other 7 ESTs did not match to the known protein coding regions. The analysis for their homology of nucleotide sequences to the genes by Blastn (Table 3-2), showed that two sequences had significant homology to S1-RNase and S9-RNase with 88% and 89% identities respectively, which indicated that the two ESTs could be S-genes and will decide GSI of Citrus grandis var. Shatinyu Hort.. Besides, some genes were also found which would take part in the SI response directly or indirectly, such as genes related to ubiquitin and 26S proteasome pathway, including proteasome subunit, ubiquitin-specific protease and forkhead box O1As; genes related to the cell signal recognition and conduction pathway, including calcium/calmodulin-dependent protein kinase, inositol 1,4,5-trisphosphate 3-kinase A and actin-binding protein; and genes related to programmed cell death, passage transportation and molecular chaperones.To make clear the biological function of these differential expressed genes abtained in the experiment, we make those ESTs with moderate to high similarity scores by Blastx to be organized into categories based on their putative function. Consequently, 9 categories were obtained according to gene ontology system. They were catalyze activity, binding function, physiology pathway, cell membrane component, molecular chaperones, transcription regulation, enzyme activity regulation, hydronium transportation and cell death (Table 3-3). Among these functions, catalyze activity takes up the largest proportion. From above, we can conclude that the SI response in Citrus grandis var. Shatinyu Hort. will be a complex process of many genes synergistic reaction.In addition, several genes were found to be related with the adversity compel of plants, virus resistance or other physiology functions, such as antifreeze protein, citrus tristeza virus resistance and dynein heavy chain family protein.All these studies lay a good foundation not only for further cloning those genes related to SI, but also for better understanding the molecular mechanism of GSI in Citrus grandis var. Shatinyu Hort..