| Upland cotton(Gossypium hirsutum L.)is the most important fiber crop in the world.Due to the conflict between food crops in the use of cultivated land and the cultivation of upland cotton,early maturity has become a key feature of short-season cotton breeding programs in China.Xinjiang is one of the largest precocious cotton ecological regions in China.There are many weather disasters in spring in northern Xinjiang,and some areas are replanted every year because of these disasters,resulting in late sowing date,high risk of cotton planting,and urgent need of early-maturing cotton varieties in production.Flower bud differentiation is an important character affecting the early maturity of short-season cotton varieties.The differentiation of fruit branches determines the amount of flowering,sexual ability and cotton yield.At the same time,flower bud differentiation appeared in the seedling stage,and the seedling stage was the most sensitive to external environmental stress and other regulatory factors.The key genes and their regulatory mechanisms controlling flower bud differentiation in cotton have not been reported so far.To study the molecular mechanism of cotton precocious regulatory network,we selected two early-maturing and two late-maturing varieties,collected shoot apexes samples before and after flower bud differentiation,and analyzed the transcriptional dynamics at seedling stage.Finally,we found a key regulatory gene that regulates the transition from vegetative growth to reproductive growth in cotton,verified its function by transgenic plants,and put forward the regulation pathway of cotton flowering.The main conclusions are as follows:1.To study the molecular mechanism of cotton precocious regulatory network,we selected two early-maturing and two late-maturing varieties growing under normal field management.The shoot apexes samples of cotyledon flattening period and 1 to 5 true leaf stage(TLS)were collected and RNA-seq.Paraffin sections of the shoot apexes showed that the flower buds of early-maturing varieties Yanzao2 and Zhongmiansuo50 appeared at the3TLS,and those of late-maturing varieties Guoxinmian你们,11 and STS458 appeared at the5TLS.The transcrioptome produced a total of 3 billion clean reads,and found a total of 49000genes expressed in at least one sample,resulting in 5312 differential expressed genes.Different expression modules are obtained by K-means cluster analysis and WGCNA analysis,and different modules play different functions.2.Cluster Ⅰ module in K-means analysis,MElightgreen module,MEtan module,MEsalmon module and MEturquoise module in WGCNA,the genes in these modules were predominantly expressed at cotyledon stage,1TLS and 2TLS in four varieties.At this time,cotton just sprouted from the soil and then began to grow.a total of 417 genes(including 33 transcription factors)were identified.The GO terms enriched in these modules were related to the transport of various nutrient elements,while the most genes related to zinc(Zn)and iron(Fe)transport were 14 and 13,respectively.These genes may play an important role in the absorption,transport and utilization of essential nutrients in cotton,and are of great significance to the normal growth,development and morphogenesis of cotton seedlings.3.In Cluster Ⅲ,we analyzed a total of 373 genes,including 55 transcription factors,and their expression increased gradually with the development of cotton seedlings.It was found that the expression of 45 GhSPLs genes increased gradually with the development stage of cotton seedlings,and the expression level of ghr-mi RNA156 in the shoot apexes was the highest in the early stage,and then decreased gradually with the extension of cotton seedling development stage.The expression trend of Gh TOE1 was opposite to that of GhSPLs,and the expression of LFY homologous gene and ghr-mi RNA172 was consistent with that of GhSPLs.At the same time,it is proved that gibberellin-related genes and trehalose pathway ultimately regulate the expression of GhSPLs.Yeast One-hybrid showed that GhSPLs could directly bind to the GTAC motif in the Gh LFY promoter and regulate the expression of Gh LFY.Finally,we propose that there is an age regulation pathway in cotton,that is,GhSPLs integrates a variety of factors to regulate cotton aging pathway.4.The MElightcyan module contains 46 genes,including 29 transcription factors.The expression pattern of these genes was consistent with flower bud differentiation,and was expressed at the 3TLS,and the expression level of these genes in two early-maturing varieties was always higher than that of two late-maturing varieties,including CAL homologous gene(GH＿D07G0876),AP1 homologous gene(GH＿A04G1749),AGL6 homologous gene(GH＿D09G0468),Gh MADS22,GHMADS23 and GhSOC1.Among them,Gh MADS23,Gh MADS22 and GhSOC1 have been proved to be involved in cotton flowering regulation.Except in Arabidopsis thaliana,CAL gene has been rarely reported in other plants.The GH＿D09G0468 that encodes the homologous gene of AGL6 and the GH＿A04G1749 that encodes the homologous gene of AP1 are named GhAGL6-D09 and GhAP1-A04,respectively.Some of them belong to ABCDE model gene,which indicates that cotton flower development may have a conservative ABCDE pattern.5.A new key regulatory gene GhCAL related to flower bud differentiation was found in the MElightcyan module.GhCAL overexpression caused Arabidopsis early flowering,whileGhCAL silencing transgenic cotton plants showed late flower bud differentiation and late flowering time.GhCAL forms heterodimer with GhAP1-A04/GhAGL6-D09 and regulates the expression of GhAP1-A04 and GhAGL6-D09.The overexpression of GhAP1-A04 and GhAGL6-D09 advanced the flowering time of Arabidopsis thaliana,while the cotton plants with GhAP1-A04 and GhAGL6-D09 silenced showed obvious late flowering.Finally,we proposed a new flowering regulation pathway mediated by GhCAL.This study clarified the molecular mechanism of cotton flowering regulation and provided good genetic resources for cotton precocious breeding. |
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