| Based on the previous studies,three cDNAs of the 14-3-3 protein family genes are cloned from the mixed anthers of wolfberry variety Ningqi No.1,named Lb14-3-3a,Lb14-3-3b and Lb14-3-3c,and bio-informatics analysis,relative quantitative expression,genetic transformation and molecular function analysis of these three genes are carried out.The specific research results are as follows:(1)The ORFs of Lb14-3-3a,Lb14-3-3b and Lb 14-3-3c genes are cloned from the mixed anthers of wolfberry variety Ningqi No.1 by RT-PCR.The ORF of Lb14-3-3a gene is 768bp,which can encode 255 amino acids with a predicted molecular mass of 61.9kD and an isoelectric point of 4.99.The ORF of Lb14-3-3b gene is 747bp long and encodes 248 amino acids with a molecular weight of 63.09kD and an isoelectric point of 4.62.Lb14-3-3c gene has a length of 777bp and encodes 258 amino acids with a molecular weight of 64.14kD and an isoelectric point of 4.95.They both belong to the 14-3-3 protein super-gene family.The α-helix is the main component of the secondary structure of these three proteins.(2)qRT-PCR analysis shows that these three anther genes are found in different tissues,organs and different anther development stages.The Lb14-3-3a gene is predominantly expressed in fruits and flowers while the transcripts in roots and leaves are low.The expression level in early anther development is relatively low.The period of sporogenous cells begins to increase,and peaks in the period of microspore mother cells.The tetrad period begins to decline and the expression level of mature pollen grains is the lowest;The expression of Lb14-3-3b gene is higher in flowers and fruits,but lower in roots,stems and leaves.The early expression of anthers is relatively low and the tetrad period begins to increase,reaching a peak during the second nuclear pollen period;The expression of Lb14-3-3c gene is the highest in stamens,and lower in roots,stems,leaves,green fruits,red fruits,pistils,petals and calyx.The expression level is the highest in microspore mother cells and the lowest in mature pollen grains.It is speculated that the family genes are involved in the regulation of the growth and development of the flower organs.(3)The plant over-expression vectors Lbl4-3-3b-pC1305.1-35S and Lbl4-3-3c-pC1305.1-35S are constructed.The plant over-expression vectors pMDC83-Lb14-3-3a and Lb14-3-3b-pC1305.1-35S constructed in the early stage of the laboratory are transferred into the tobacco variety"Sanshengyan"by the Agrobacterium tumefaciens-mediated pathway to obtain positive plants.The results show that 6 positive transgenic plants are obtained,the positive conversion rate was 54.5%and the growth rate of Lb14-3-3a tobacco plants is faster,and 4 flower organs are distorted.And there are 6 symmetrical petal petals,and the stigma is higher than the stamen.This is consistent with the stigma and anther characteristics of the male sterile mutant Ningqi No.5.;The results show that 9 positive Lb14-3-3b tobacco plants are obtained,the positive conversion rate reached 75%,the growth rate of Lb14-3-3b tobacco plants is slower,there are 5 flower organs are distorted,And there is a loss of petals,resulting in the whole flower form into a four-cornered shape,lacking one filament and the anther development is incomplete,making it difficult for tobacco plants to complete normal sexual reproduction.It is speculated that the Lb14-3-3a and Lb14-3-3b genes may affect the growth and development of tobacco plants,and play a certain regulatory role in the abortion process of pollen.The results of this study provide insights and theoretical support for the in-depth study of the molecular mechanism of Lb 14-3-3 protein on anther and pollen development.The results provide a theoretical reference for further study on the regulation of Lb 14-3-3 protein family genes on the growth and development of scorpion organs and sorghum plants in Lycium barbarum L.At the same time,the results also provide a new idea for expanding the regulation mechanism and molecular mechanism of Lb 14-3-3 protein in plant growth and development. |
