The Basic Studies On Sexual Determination And Related Gene In Ramie (Boehmeria Nivea)

Rarnie(Boehmeria nivea (L.) Gaud.) is perennial bast fiber crop. Scince ramie is a cross-pollinatedcultivated plant with complex genetic background, its offspring can be badly segregated. It was moreexpensive that the male flower must be deleted in heterosis and breeding by clone method. To solvethese problem, it is very important to study on sexual determination and related genes to control ramiesexual expression by researching bud morphology and differentiation, physiology and moleculargenetics in sexual special ramie. This research could promote to ulitize the heterosis and improve on thebreeding method.About the studis on sexual determination of ramie, the gynandrous primordium was formed firstlyduring sex differentiation in male flower. Then the male flower could be shaped by degenerated thegynoecium. But the androecium was not appeared in female flower. When the bud growed to 0.5centimeter before the the gynandrous primordium was formed, the male were easily induced by GA₃among apomixes species. The exact time and maker of external morphology that happened at sexualdetermination date in ramie were not known.The study of relation between the exogenous hormone andsex was mostly focus on reversing sex in ramie.It was not reported about the related genes of sexdetermination in ramie.In this paper, the ramie materials with unusual sex was studied on observing in botany character,researching bud differentiation, mensurating endogenesis ethylene content, spraying the ethyleneinhibitor, cloning the ACC synthase and studying spatio-temporal expression.The main result of thispaper are as follows:1. The female ramie (GBN-09) and hermaphrodite ramie(GBN-08) were investigated in second crop.There are light red leafstalk, connate stipul, kelly stem, green bud and small stem spot in GBN-08.it was different with GBN-09 that have green leafstalk, asunder stipul, white yellow bud and bigstem spot.2. The growth trend was obtained by observing the GBN-09 and GBN-O8.The result showed the daygrowth trends of stem highness was parabola on GBN-08 and GBN-09 in the second crops, wasgradually reduced in third crops. The day growth trends of node numebers was faster ininitiat-stage than late-stage. It is the same growth trends between the stem highness and leaf lengthand leaf width, and the growth trends is positive correlation between the leaf length and leaf width.3. The characteristic of flowering were studied in female ramie (GBN-09) and hermaphrodite ramie(GBN-08). The result showed that the sex of individual plant of ramie was determined by the fastbud (which was earliest differentiation). If the fast bud was female, that the individual plant oframie was female. But if the the first bud was male, that the individual plant of ramie washermaphrodite.4. Morphological observation of flower bud differentiation were done with GBN-08 and GBN-09 byparaffin section. It was the time of sex determination in hermaphrodite ramie including 28-30 node When the bud was 0.2±0.067 centimeter; And It was the time of sex determination in female ramieincluding 24-29 node When the bud was 0.25±0.037 centimeter.5. The ethylene release rate was measured in shoot tip of different sex ramie in different time, the budof same node in female and hermaphrodite ramie in second crops and different sex singleinflorescence in hermaphrodite ramie. The result indicated there was closed relationship betweensexual different ramie and ethylene release rate. The female ramie could be induced by highethylene release rate. the ethylene content in ramie plant with female flower is higher than ramieplant with androgynary in stem apex, leaf and flower. In flower organ, the ethylene content ishigher in female flower than male flower and mixed inflorescence. The sex expression of ramiewas found to be controlled by foliar spray with 2-aminoethoxyvinylglycine(AVG) and AgNO3.AVG at a concentration of 300,100mg/L and AgNO3 at a 100, 300,500mg/L caused increase ofmaleness and inhibition of fe-male flower. AVG at a concentration of 300mg/L were the best.6. It is necessary to extract total RNA from plant tissue.A new reagent—RNAplant was screened outto extract RNA from ramie leaf, stern, female flower and fruit by used five different reagents.RNAswere effective extracted and the high quality RNA can be used for cDNA synthesis, RT-PCR andothers downstream experiment.7. ACC synthase(ACS) was a key enzyme for ethylene biosynthesis. In this paper, four degenerateoligonucleotide primers were designed, coding for consernative amino acid regions in ACCsynthase protein family of dicotyledon. Reverse transcription PCR and Nest PCR were performedon total RNA extracted from shoot tip of ramie, and produced 615bp fragment. By using theprogram of BLASTn and BLASTx on NCBI GenBank database, the fragment was ACC synthasegene, coded 205 amino acids. Domain of deduced ACS amino acids in ramie was aligned withACS amino acids of twenty plant. The result showed that there are five conserve domain in ACSamino acids of ramie.The ramie have higher similar with soybean by phylogeny.8. To study the differential expression of ACS gene at different development stage and in differenttissues of ramie,relatively quantitative analysis was performed by Realtime-PCR. The expressionlevel of ACS gene in stem apex was higher than leaf, stem and lowest in root in seedling. Theexpression level of ACS gene was lower in anthesis and fruiting period. The expression level ofACS gene in stem apex or bud of GBN-09 was higher than that of GBN-08 when size of the firstbud was 0.75 centimeter.So, the new progress about the sex determination of ramie was obtained in botany character,researching bud differentiation, mensurating endogenesis ethylene content, spraying the ethyleneinhibitor, cloning the ACC synthase and studying spatio-temporal expression.. The results provided aexprerimental and theoretical basis for controling the sex of ramie, utilizing the hybrid vigor andimproving the breeding methods.