Cloning And Functional Analysis Of The Auxin Internalization Gene LAX3 In Guizimai

Auxin plays an important role in regulating the growth and development of wheat and its response to stress,and these regulatory processes all depend on the establishment of auxin concentration gradients.As an important feature of auxin,polar transport is a key factor in establishing auxin concentration gradients.Therefore,cloning and studying the auxin infusion gene in wheat is of great significance to understand the physiological process and mechanism of its involvement in auxin-mediated wheat growth and development.In this study,the bioinformatics and expression pattern analysis of Ta LAX3-1B gene encoding wheat auxin internal transport protein was carried out,and the biological function of Ta LAX3-1B gene was explored by overexpressing transgenic tobacco and rice.The main findings obtained are as follows:1.Bioinformatics analysis of Ta LAX3-1B,an auxin infusion gene of noble purple wheat,showed that the conserved domain of Ta LAX3-1B protein has a specific site of PLN03074,and the PLN03074 family proteins encode auxin permease,which has the function of promoting auxin.Introvert function.The coding region(CDS)of Ta LAX3-1B gene is 1587 bp in length and encodes 528 amino acids.The phylogenetic relationship results showed that the Ta LAX3-1B gene of ’Guizimai No.1’ was most closely related to wild emmer(Triticum dicoccoides),barley(Hordeum vulgare)and oil palm(Elaeis guineensis),with more than 90% of the sequences.similarity.2.Subcellular localization analysis showed that Ta LAX3-1B protein was located on the plasma membrane,and it was also found that talax3-1b protein was expressed on the guard cell membrane.The tissue expression specificity analysis of Ta LAX3-1B showed that Ta LAX3-1B gene was expressed in the roots,stems,leaves and mature seeds of transgenic lines,with the highest expression in leaves and mature seeds,followed by the expression in roots and the lowest expression in stems.At the same time,GUS histochemical staining showed that Ta LAX3-1B was mainly expressed in the veins and stomata of leaves.In taproot,it was expressed in the meristematic zone and mature zone of taproot,and micro-expression was also detected in the meristematic zone of lateral root.At the same time,it is expressed in pollen.3.The stomatal movement experiment found that under normal conditions,the width of the stomata in the lower epidermis of the transgenic line was significantly smaller than that of the wild-type plant.The pore length and pore width are opposite.Under ABA treatment,we found that ABA promoted stomatal closure,and there was no significant difference in stomatal morphology between transgenic lines and WT lines after treatment.At the same time,the stomatal morphological changes of the transgenic lines were smaller than those of the wild type,and under the ABA treatment,the stomata of the transgenic lines did not close normally,indicating that the transgenic lines were less sensitive to ABA treatment than the wild type.In addition,the leaf water loss rate of the overexpression lines was significantly reduced.These results suggest that Ta LAX3-1B overexpression affects stomatal morphology and keeps stomata relatively closed.4.Under normal conditions,Ta LAX3-1B overexpression increased the contents of IAA and ABA in leaves of tobacco overexpression lines,and the transcription levels of IAA and ABA biosynthesis-related genes were significantly up-regulated,indicating that Ta LAX3-1B was involved in the IAA and ABA signaling pathways.At the same time,the expression levels of IAA and ABA-mediated stomatal closure genes ABP1,PYL2 and SLCA1 were also significantly up-regulated.Based on the above results,Ta LAX3-1B promotes the accumulation of IAA and ABA by participating in the IAA and ABA signaling pathways,while the expression of IAA and ABA promotes the accumulation of IAA and ABA.Accumulation induces the expression of stomatal closure-related genes such as ABP1,PYL2,and SLCA1,which together promote stomatal closure.5.Under 300 m M salt stress,tobacco overexpression lines showed stronger salt tolerance phenotype than WT.Through the analysis of physiological indexes of WT and tobacco over-expression strains under salt stress,it was found that after salt treatment,over-expression strains had higher salt tolerance,in which proline content in over-expression strains was significantly higher than that in WT,while malondialdehyde content was significantly lower than that in WT.At the same time,the overexpressed transgenic strain of Ta LAX3-1B showed the phenotype of reducing the accumulation of reactive oxygen species(ROS)and increasing the activity of antioxidant enzymes.6.In transgenic rice,we found that the overexpression of Ta LAX3-1B increased the accumulation of anthocyanins in rice leaves,while the transcription levels of structural genes ANS,CHI and F3’H in anthocyanin biosynthesis pathway were significantly up-regulated,indicating that Ta LAX3-1B was involved in the signal pathway of anthocyanin synthesis.To sum up,the research in transgenic tobacco shows that wheat Ta LAX3-1B can increase the content of endogenous IAA and ABA,which leads to the decrease of leaf stomatal aperture and water loss rate,and plays an important role in stomatal closure.On the other hand,Ta LAX3-1B can reduce the accumulation of reactive oxygen species and increase the activity of antioxidant enzymes,thus improving the tolerance of plants to stress,which plays an important role in plant stress resistance.Studies in transgenic rice show that wheat Ta LAX3-1B can promote anthocyanin accumulation and play an important role in anthocyanin synthesis signal pathway.The results of this study provide an important theoretical basis for exploring the mechanism of Ta LAX3-1B gene regulating wheat growth and stress resistance.