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Study on Basic Biology of Elongated Uppermost Internode (eui) Gene Expression of Rice

Author: XiaoHuiHai
Tutor: ChenLiangBi
School: Hunan Agricultural University
Course: Botany
Keywords: thermo-sensitive male sterile(TGMS) rice temperature elongated-uppermost-internode(eui) specifc expression suppression subtractive hybridization (SSH) cell
CLC: S511
Type: PhD thesis
Year: 2005
Downloads: 126
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Abstract


The poor exserted panicle of the male sterile line in rice is one ofmain problems in hybrid seed production. In order to overcome the panicleenclosed, spraying GA3 is the only sure to make panicle exserted. However,elongated uppermost internode(eui) gene is very useful for seedproduction of hybrid rice. Changxuan 3S was thermo-sensitive malesterile(TGMS) rice with eui gene selected from seeds of Peiai 64Sirradiated by 350 Gy60Coγ-ray. To provide science evidence and theorysupport for exploiting and utilizing TGMS rice Changxuan 3S with eui gene,the correlativity between panicle exsertion of Changxuan 3S and naturetemperature was analyzed, and effect of temperature on eui gene expressionwas studied, and the cell number and length were researched. At the sametime, specifc expression cDNA seqence of the uppermost internode forchangxuan 3S were isolated and cloned by using supperssion subtractivehybridization (SSH). The main results were showed as below:The culm length of Changxuan 3S was 24.3 cm longer than that of Peiai64S, the increased culm length for Changxuan 3S was mainly due to theelongation of the uppermost internode. The length of the uppermostinternode for Changxuan 3S was 16.6cm longer than that of Peiai 64S, andthe panicle exsertion was 13.8cm longer than that of Peiai 64S at naturaltemperature condition. Cytological studies on Changxuan 3S and Peiai 64Sshowed that the number of external and internal parenchyma cells in theuppermost internode of Changxuan 3S were 1 248 and 580 more than thoseof Peiai 64S respectively and the length 23.3μm and 38.3μm longer respectively. The number and length of cell had no obvious difference inbasal and top sections, but the length of parenchyma cell in middle sectionof the uppermost internode in Changxuan 3S was much longer than that inPeiai 64S, i.e. the two kinds of parenchyma cells were 24.8μm and 48.7μm longer respectively. Those resulted in 16.6 cm longer for uppermostinternode and 13.8 cm longer for panicle exserted in Changxuan 3S thanthat in Peiai 64S. It is, therefore, evident that elongation of uppermostinternode of Changxuan 3S is due to the increase of cell number and cellelongation. And the elongation of later was more significant.The length of panicle exsertion of Changxuan 3S was negativelycorrelated with a daily average temperature during 20-9d beforeflowering and with a most effective period between 17-12d at naturaltemperature condition.The optimal panicle exsertion of Changxuan 3S was recorded in thisstage of 17-12d before flowering when treated for 6 d at 24℃. The resultof artificial temperature treatment was the same as the correlationanalysis between panicle exsertion and daily mean temperature of 17-12dbefore flowering at natural temperature condition, i.e. the criticalperiod of eui gene expression to temperature was the period of preblooming17-12d (forming pollen mother cell and meiosis)Changes in length of elongated uppermost internodes under thecondition of constant 24℃showed that the internodes began elongation atpreblooming 12d. At preblooming 8d, high elongation of the internodes wasrecorded, and the elongation rate of internodes for Changxuan 3S wasfaster than that of Peiai 64S. The fastest period of internode elongationof Changxuan 3S was the fourth day preblooming to 0 day (flowering) withthe rate of 5.8 cm per day, which contributed 63.38%of the total length,while Peiai 64S had 2.65 cm elongation per day and 50.72%of the totallength. The internodes elongation rate decreased obviously afterflowering, and stopped elongation at the third day after flowering. The results demonstrated that the difference of internodes elongation betweenChangxuan 3S and Peiai 64S was mainly the fourth day preblooming toflowering and the mean elongation rate of internodes for Changxua3S was2.2 times faster than that of Peiai 64S.When the temperature treatments at 22℃, 24℃, 26℃or 28℃wereimposed on Changxuan 3S during the most sensitive period of eui geneexpression, the expression of eui gene was depressed under the temperatureof 28℃, whereas improved at the temperature of 22℃, 24℃, 26℃, in thatthe lower the temperature, the longer the panicle exsertion. Afterdifferent temperature treatments, the cell number and mean length ofelongated uppermost internodes were lower along with increasingtemperatures in Changxuan 3S. After treated at 28℃, the number ofoutermost and innermost parenchyma cells was 771 and 292 respectively morethan those of 22℃. The mean length of cells was 13.9μm and 24.6μmrespectively shorter than those of 22℃. The mean length of the two cellshad no obvious difference in basal and top sections, but the mean lengthof outermost and innermost parenchyma cells was 18.0μm and 35.4μmrespectively shorter than those of 22℃in middle sectionsA subtracted cDNA library specific to Changxuan 3S was constructedby SSH and pGEM-T Easy Vector cloning, which consisted of 130 individualrecombinant clones. Each clone had an insert with the average size of 185bp, and the frequency of inserting was 100%.Using PCR-select differential screening kit, the 96 recombinantclones of the constructed above cDNA library were chosen randomly and thenhybridized with forward and reverse subtracted probes and unsubtractedprobes. The results showed that 20 positive clones specific to theelongated uppermost iternode of Changxuan 3S were obtained. To confirmtheir positive results, 8 clones of them were chosen randomly from theabove positive clones and analyzed by Northern blot hybridization. Theresults showed that the probe built from 1 candidate clone hybridized only with Changxuan 3S mRNA, 4 clones hybridized with Changxuan 3S and Peiai64S mRNA, while the other 3 clones had no hybridization signals withChangxuan 3S and Peiai 64S mRNA, which probably represented cDNA clonesexpressed by low-abundance transcripts in Changxuan 3S.The positive clone specific to the elongated uppermost iternode ofChangxuan 3S was sequenced. The relationship between the gene expressionand elongation of the uppermost internode for Changxuan 3S was discussed.

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