| With the development of human society and the increase of human production activities,all kinds of ecological environmental problems have become increasingly prominent.In the Yellow Sea of China,the green tides of Ulva prolifera have been breaking out for 13 years,which not only threaten the ecological health and safety of the surrounding waters,but also bring huge economic losses to the aquaculture and tourism industry of coastal cities.How to prevent and control the green tides of U.prolifera scientifically and effectively is the primary problem at present.At present,there are some preliminary research results on the green tides of U.proliera in the Yellow Sea:it was initially formed in the laver culture area of the Subei Shoal,which contains a large number of microscopic propagules of Ulva,becoming the "seed bank.of the continuous outbreak of the green tides of U.prolifera in the Yellow Sea;at the same time,compared with other green algae,U.prolifera has more efficient nutrient absorption capacity,which provides the basis for U.prolifera to grow and breakout in a short period of time.However,the molecular mechanism of the growth and development of U.prolifera in the Yellow Sea has not been reported.This study focuses on the molecular mechanism of the growth and development of U.prolifera in the Yellow Sea:First of all,we organized two provenance surveys to collect the sediments of laver culture area in the Subei Shoal.Through culture,we found that there were microscopic propagules of green macroalgae in the sediments,including Ulva and Blidingia and other algae,among which Ulva was the most abundant.Meanwhile,Ulva included U.flexuosa,U.prolifera and U.compressa And U.linza,in which the number of U.flexuosa is the most.This result improves the species diversity of green algae's microscopic propagules in laver culture area of the Subei Shoal,and provides a basis for us to further understand the ecological process of the outbreak of the U.prolifera green tides in the Yellow Sea.Secondly,in order to investigate the dominant position of U.prolifera in the Yellow Sea green tides,we analyzed the comparative transcriptome sequencing of the above four Ulva specie.The sequencing results showed that there were a group of core genes with similar number in four species,and these genes were involved in more than half of the algae metabolism.Meanwhile,compared with other three Ulva,many genes related to growth and development of U.prolifera have been enriched,such as PK and Nrt,which may make U.prolifera more competitive in growth and become the dominant species of the Yellow Sea green tides.This result provides us with a way to understand the molecular mechanism of U.prolifera becoming the dominant species in the Yellow Sea green tides.Third,the above studies on the growth and development characteristics of U.prolifera are based on the coexistence of U.prolifera and bacteria,whether U.prolifera can also erupt into green tides under sterile conditions is unknown.In order to study the growth characteristics of U.prolifera under aseptic condition,we successfully constructed the callus of U.prolifera by using 5 kinds of antibiotics.The results showed that under aseptic condition,the sporophyte/gametophyte of U.prolifera could not complete the normal morphogenesis and growth.The successful construction of callus of U.prolifera laid a foundation for further study on the morphogenesis and growth of U.prolifera.Fourth,we studied the induction of callus of U.prolifera by four epiphytes.According to the study of 16 strains of U.prolifera isolated in our laboratory,it was found that four strains of bacteria had obvious induction effect on the callus of U.prolifera,which were E68(Roseobacter sp.),E69(Marivita sp.),E104(Bacillus sp.),E110(Maribacter sp.).According to the experiment of inducing callus of U.prolifera by 4 strains of bacteria,it was found that 4 strains of bacteria need to reach a certain working concentration in order to play the induction role.At the same time,the induction role of different bacteria in the same working concentration is also different,among which E110 bacteria has the best induction ability.In addition,4 strains of bacteria had no necessary to contact with U.prolifera callus directly to induce it.The results of transcriptome sequencing of callus and bacterial induced callus showed that a large number of genes related to growth and development were differentially expressed,which laid a preliminary foundation for elucidating the molecular mechanism of morphological formation and growth and development of U.prolifera induced by bacteria.Meanwhile,thallusin,which can induce other Ulva,such as Monostroma oxyspermum,can also induce the morphogenesis and growth of U.prolifera callus,which provides a way for us to identify the active substances of bacteria.Finally,in order to further study the molecular mechanism of the growth and development of U.prolifera,especially the molecular mechanism of carbon fixation pathway,we selected carbonic anhydrase(CA),a key enzyme essential to the carbon fixation pathway of green algae,as the research object.We cloned three carbonic anhydrase genes of U.prolifera:Up?CA1,UpyCA2 and Up?CA1.Bioinformatics analysis confirmed that Up?CA1 and UpyCA2 belong to the family of ?-CA family,and UpaCA1 belongs to the family of ?-CA family.Phylogenetic tree analysis showed that they have species conservation in evolution.The overexpression vector of UpaCA1 gene was constructed successfully and UpaCA1 was an active protein.The qRT-PCR results showed that the three carbonic anhydrases were sensitive to high temperature,low temperature,high salt,low salt,high light and low light conditions,and were inhibited by these conditions,which was consistent with the inhibition of the growth of U.prolifera under these conditions,indicating that environmental factors may directly regulate the growth of U.prolifera by regulating carbonic anhydrases.The three carbonic anhydrases adapted well to the pH 7.5-8.5 culture conditions,and their transcription level was in a dynamic equilibrium state for a long time.The results provide a molecular basis for the study of carbon fixation pathway and growth rule of U.prolifera under different conditions.For the first time,the following conclusions are proposed in this study:1.For the first time,we analyzed the differences of transcription level of four Ulva:U.flexuosa,U.prolifera and U.compressa And U.linza.The results showed that compared with the other three Ulva species,U.prolifera enriched many important genes related to growth and development,which provided a research basis for understanding U.prolifera as a dominant species of green tides in the Yellow Sea from the perspective of molecular evolution.2.The callus of U.prolifera was constructed for the first time,which indicated that U.prolifera could not complete normal morphogenesis and growth and development under sterile condition,which provided a basis for the study of the molecular mechanism of morphogenesis and growth and development of U.prolifera.3.For the first time,the induction characteristics of four kinds of bacteria on U.prolifera callus were investigated,including different bacteria,different concentrations of the same bacteria have different induction effects on the callus of U.prolifera,bacteria do not need to contact with the callus directly to perform the induction.The molecular mechanism of U.prolifera callus induced by bacteria was preliminarily investigated by transcriptome sequencing.It was found that the induction of U.proljfera callus by bacteria caused a large number of growth and differentiation related genes expression differences.4.For the first time,thallusin was found to induce callus of U.prolifera.It provides a way to further determine the specific substances and mechanism of the callus induction of U.prolifera by bacteria.5.The carbonic anhydrase gene of U.prolifera was cloned and analyzed for the first time.It is also the first carbonic anhydrase gene obtained from Ulvaceae.Three carbonic anhydrase genes were obtained:UpyCAl,UpyCA2 and Up?CA1.The results of activity test showed that Up?CA1 was an active protein.At the same time,qRT-PCR results showed that the three carbonic anhydrases had different response to different environmental conditions,which provided the basis for understanding the molecular mechanism of the growth and development of U.prolifera,especially the carbon fixation pathway. |
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