Molecular Cloning And Expression Analysis Of The Genes Differentially Expressed In A Rice Hybrid And Its Parental Lines

The explosion of world population spurs a need for more food supply. The popularization of hybrid varieties plays a major role in the increase of crops productions in the past few decades. Although utilization of heterosis has achieved great success, the mechanism underlying this phenomenon remains poorly understand. In turn, it limits the full utilization of heterosis. This dilemma plus the environmental stresses and disasters urgently call for further understanding of heterosis. It is important to isolates the genes differently expressed in hybrid and parental lines, because it is necessary for understanding: 1) the gene expression pattern in hybrid and its parents; 2) gene functions and 3) mechanism of heterosis. Rice is the most important staple food in China. As a model species, it has received extensive attention in recent years. Its significance together with available genomic information and mature transformation system makes it an ideal species in exploration of heterosis. In our experiments, we have analyzed the gene expression pattern in an excellent HL-type rice hybrid and its parents, and isolated a large number of differentially expressed genes. Among them, two genes encoding key enzymes in the Calvin cycle were particularly investigated. The results are listed below.1. cDNA-AFLP was employed to compare the gene expression patterns and to isolate the differentially expressed genes between a rice hybrid and its parents. Rice leaves at tillering stage and flowering stage, and panicles at uninuclear- and binuclear stages were used as materials. Totally, around 2720 genes were detected in each sample by using 64 primer combinations. The gene detected in each sample accounts for -5% of total rice genes. Both qualitative and quantitatively differences in gene expression were found between hybrid and parental lines of each developmental stage or tissue. The former kind of differentially expressed genes can be divided into: 1) BP type, i.e., the genes expressed in both parents, but not in hybrid; 2) FS type, .i.e., the genes only expressed in hybrid but not in parents; 3) UP type, i.e., the genes only expressed in one parent, and not in hybrid or another parent; and 4) UPF, i.e., the genes silenced in one parent, but expressed in hybrid and another parent. Thelatter kind of differentially expressed genes can be divided into: 1) IF, i.e., stronger expression in hybrid, but weaker in both parents; 2) IP, i.e., stronger expression in both parents, but weaker in hybrid; 3) IFP, that is, stronger expression in hybrid and one parent, but weaker in another parent; and 4) IUP, stronger expression in one parent, but weaker in hybrid and another parent. 257 differentially expressed cDNA fragments were isolated in total, accounting for 9.448% of all detected genes. Among them, there are 80 genes that only expressed in hybrid, 36 genes that display stronger expression in hybrid compared to parents, 90 genes that specifically expressed in parents and 51 genes that show stronger expression in parents compared to hybrid. Each of the types accounts for 31.13%, 14%, 35.03% and 19.84% in total differentially expressed genes, respectively. The average proportion of genes that show specifically expressed or stronger expression in each parent is 17.51% and 9.92%, respectively. It is obviously lower than that of hybrid, especially the specifically expressed genes. 197 differentially expressed cDNA fragments were recovered and 55 of them were sequenced. 47 of sequenced fragments are cDNA, and others are DNA contamination or rRNA. BLAST results indicate that among the cDNAs, 1) 12 genes (occupy 25.5%) are involved in metabolism, including seven genes related to photosynthesis; 2) four genes (occupy 8.5%) were involved in gene regulation or mRNA translation, including three transcription factors; 3) three genes (6.4%) are implicated in resistance (UV or disease), and equal number genes are related to cell structure; 4) one gene (2.2%) MAPK is involved in signal transduction, and the remained 24 genes (51%) are functionally unknown. These results suggest that the genes related to photosynthesis and the transcription factors involved in gene regulation may play pivotal roles in heterosis. The sequenced genes were also mapped on rice chromosome. The results revealed that these genes are widespread on all the chromosomes, but the gene distributed on Chr.l, 2, 3,4 and 9 is obviously more than on other chromosomes. Compared to previous QTLs mapping, we proposed that the genes mapped on Chr. 1 and 3 are more likely related to heterosis.2. As described above, the genes involved in photosynthesis may play important roles in heterosis. So, the two Calvin cycle genes were particularly investigated. Based on sequence of P400, we cloned a 1663 bp full-length cDNA (OsPrk) encoding rice PRKase by RACE. OsPrk is composed of 165bp of 5' untranslated region, 1212 bp of open reading frame, 284bp of 3' untranslated region and a poly(A)n tail. The corresponding genomic DNA is 2443 bp, containing five exons and four introns. The gene structure is very similar to wheat PRKase gene. Southern blot result revealed that OsPrk is single locus in the rice genome. It is localized in the region 28.32-28.33 Mb on Chr.2, and flanked by markers RM450 and MRG0168. RT-PCR analysis indicated that it is expressed in all the tissues we tested, i.e., root,stem, leaf, bud, panicle and anther. Its transcription is dramatically activated by light illumination in ten minutes. Northern blot results showed that transcript level of OsPrk is lower in female parent, higher in hybrid and highest in male parent at both tillering and flowering stages. Our results also demonstrated that the expression of OsPrk is modulated by plant hormones and environmental stresses. NaCl, ABA, MeJA or glucose treatments resulted in down-regulation of transcript level of OsPrk gene, whereas, externally applied GA led to up-regulation of OsPrk transcript level. Interestingly, glucose treatment can down-regulates OsPrk transcript level, but didn't affect PRKase activity. It is firstly reported that the Calvin cycle gene is regulated by several physiological stresses at multiple levels. Biochemical assay showed that pH 7.8-7.9, Mg2+ density 12-15 mM is optimal condition for rice PRKase activity, and 40mM NADP+ can completely repress the activity of rice PRKase.3. Another Calvin cycle gene OsSbp encoding rice SBPase was also isolated by RACE. It is composed of 1566 bp, in which a 1179 bp open reading frame encoding 392 AAs peptide is included. The corresponding genomic DNA is 2594 bp, containing eight exons and seven introns. OsSbp gene is single locus in the rice genome. It is localized in the region 8.42-8.59 Mb on Chr.4, and flanked by markers S20654 and RZ602. OsSbp is expressed in all the tissues we examined, including root, stem, leaf, bud, panicle and anther. Its transcription is also activated by light illumination in ten minutes. Sequence analysis suggested that the light-regulated sequence of OsSbp gene exists in the 1220 bp fragment at 5' upstream from translation start codon. Some hormone- and stress-responsive motifs (such as, Sbp-CMAla, RbcS-CMA7a, HSE, ABRE, AuxRR-Core, TATCCAT-box and CGTCA) were found existing in this fragment, suggesting OsSbp could be regulated by these stimuli. Our results showed that exogenous NaCl, ABA, MeJA or glucose could down-regulate OsSbp transcript level, whereas GA could up-regulate its transcript level. These results demonstrate that OsSbp gene can be regulated by plant hormones and environmental stresses at multiple levels. This finding is consistent with the regulatory characters of OsPrk gene, suggesting this phenomenon may be commonplace to the Calvin cycle genes, but not particular to OsPrk and OsSbp genes. To test this supposition, 5'upstream sequence of available Calvin cycle genes were compared. The results revealed that most of the Calvin cycle genes contain hormone-and stress-responsive elements, such as, ABRE, ERE.TCA-element, TGACG, C-repeat DRE, HSE.WUN and chs-cma2a motifs. These findings confirm our supposition and suggest that Calvin cycle genes are potentially regulated by a lot of environmental and physiological stresses.