| Nelumbo nucifera Gaern is one of the top ten famous flowers in China and has a high ornamental,economic and cultural value.In the low light condition,the lotus flower bud is easy to abort and cannot bloom,which greatly affects its wide application and industrial value.The weak light of the duration causes the growth and development of the plant to be affected,and the most direct effect on the plant is to cause a decrease in photosynthetic efficiency and a decrease in carbohydrate synthesis.In the process of flower bud differentiation and flower organ formation and growth,it is necessary to consume a large amount of nutrients,among which sugar substances are particularly important for the formation of flower buds.One of the important pathways in sugar metabolism is the trehalose pathway,in which trehalose-6-phosphate regulates plant flowering,and trehalose-6-phosphate synthase is a key enzyme in the synthesis of trehalose-6-phosphate.Plants play an important role in germ development and flowering regulation.At present,the research on molecular breeding of lotus is still in its infancy,and the cause of flower bud abortion under low light has not been reported.In this study,the lotus flower seedlings’Xiaguang Dingzhi’,’Hongqi Tuzhu’ and ’Xuelian’ were used as test materials,and the growth index,flowering quality,photosynthetic index and soluble sugar content were measured and compared by setting shading treatment.The abundance of flower buds was observed and the apoptosis was detected.The key genes in response to low light and their expression characteristics under different stresses were screened by transcriptome,and TPS1(trehalose-6-phosphate synthase 1)and SnRK1 in lotus((Sucrose-non-fermentingl-related kinasel)Gene cloning and sequence analysis,construction of NnTPS1 gene overexpression vector to Arabidopsis for heterologous expression,complementation test of yeast TPS mutant,aiming to explore lotus flower bud under low light The cause of abortion and related mechanisms.The main findings of this experiment are as follows:1 Physiological response of lotus under shading conditionsCompared with the whole day,the shading treatment forced the growth of the lotus flower to be blocked,the flowering amount decreased significantly,and the flowering period was delayed and shortened.The photosynthetic characteristics analysis showed that the light compensation points of the three varieties decreased,and the light compensation point of the ’Xuelian’ variety had the largest decline;The content of soluble sugar decreased under light,and the sucrose content in rhizomes was the most affected by shading,which decreased by 5.3 times,4.8 times and 2.4 times respectively.The total amount of chlorophyll increased,and the total amount and growth of ’Snow Love’ was the most significant.The total amount after shading was 6.878±0.959a mg·g-1,an increase of 20.5%.Correlation analysis showed that the flowering amount of lotus flower was significantly positively correlated with the light compensation point,total sugar content and leaf number of lotu.2 Tissue section observation of aborted flower budsBy observing the flower bud slices of lotus abortion,it was found that the flower stalks appeared to be separated from the normal flower buds,and the petal primordia showed a shrinking dry state.The stamen primordia showed a disintegrated fracture state,while the carpel primordia The basal part also showed the characteristics of fault developmental malformation,which indicated that the flower bud stayed in the carpel and flower receptacle of the lotus flower bud to continue the differentiation stage,and then terminated development and went to aging.Through tunnel staining technology,it was found that the number of apoptotic flower buds was significantly increased compared with that of normal flower bud cells,and it was confirmed that apoptosis was the main cause of flower bud abortion caused by weak light.3 Screening and functional analysis of key genes in response to low lightTranscriptomic analysis screened for two key genes in response to low light,NnTPSl and NnSnRK1.The results of weak light treatment showed that NnTPS1 and NnSnRK1 strongly responded to weak light stress such as shading and flooding,and there was antagonistic effect between them.The quantitative results of sucrose treatment indicated that the expression of NnTPSl increased with the increase of sucrose concentration in a certain sucrose concentration.The NnTPSl and NnSnRK1 genes were successfully cloned from lotus flowers.The ORFs of the two genes were 2802 bp and 1527 bp,respectively,which were closely related to Arabidopsis and rice.The pFastR05-NnTPS1 overexpression vector was constructed,and Arabidopsis thaliana was heterologously expressed.The transgenic lines overexpressing NnTPS1 were obtained by using RT-PCR identification technology.The preliminary results showed that Arabidopsis thaliana overexpressing NnTPS1 compared with wild has stronger resistance.Using the complementation experiment of yeast TPS mutant strain,it was proved that NnTPS1 can complement the TPS function of yeast mutant strain and prove that it is a functional TPS gene. |
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