Screening Differentially Expressed Genes Between Soft-endocarp And Hard-endocarp Hawthorns And Cloning And Identification Of Transcription Factors Regulating Lignin Biosynthesis

Hawthorn (Crataegus spp.) is the plant of Rosaceae. It is an important feature of lignified-endocarp. Soft-endocarp hawthorn is a unique and precious resource in china. The most prominent characteristic is a half lignified-endocarp, which can eat with the consumption of flesh. In order to explore the development and formation mechanism of soft-endocarp, soft-endocarp hawthorn’Kaiyuanruanzi’and hard-endocarp hawthorn ’Chuizhishanlihong’are used as materials and several approaches are employed. The endocarp of lignin deposition process is observed by Wiesner staining; Differentially expressed genes are analyzed by high-throughput transcriptome sequencing, and verified by qRT-PCR in soft-endocarp and hard-endocarp hawthorns; We have cloned two genes, containing one NAC transcription factor and one MYB transcription factor relating to lignin biosynthesis, their encoding sequences are analyzed, their function are verified. Main results and conclusions are as follows.1. Soft-endocarp hawthorn’Kaiyuanruanzi’ and hard-endocarp hawthorn ’Chuizhishanlihong’are used to observe the situation of lignin deposition by Wiesner staining. The result showed that endocarp layers of’Kaiyuanruanzi’ slightly turned red33days after bloom. They are obviously dyed red43days after bloom. Soft-endocarp hawthorn can always be cut with a knife because it has soft endocarp layers. The endocarp layers of ’Chuizhishanlihong’slightly turned red23days after bloom. Then, endocarp layers are significantly thickened. The endocarp layers almost all dyed red35days after bloom are completely hardened and could not be cut with a knife. Based on the observation results, we conclude that lignin of hard-endocarp hawthorn substantially deposited23days after bloom.2. Total RNA is isolated from fruits that23days after bloom of soft-endocarp hawthorn and hard-endocarp hawthorn, they are used as the construction of cDNA library. We have obtained14659624and11538395pieces of high-quality short Read by Illumina high-throughput sequencing technique, respectively. Moreover,41723and39663pieces Uingene with an average length of703bp and656bp are obtained by Trinity after de novo assembly of short Read. Accordingly, a high-quality transcriptome database of hawthorns has been constructed successfully.3. The result of Unigene expression analysis show the amount of commented for lignin biosynthesis enzyme ecoding genes, C4H, HCT, C3H, CCR, CCoA-OMT, F5H and CAD, four NAC transcription factor genes and twteen MYB transcription factor genes are significantly down-regulated in soft-endocarp hawthorn. We validate these genes differentially expressed by qRT-PCR. The result of qRT-PCR is consistent with the result of transcriptome data. These findings suggest that lignin biosynthesis relating to transcription factors are expressed lower level significantly, it cloud be the molecular mechanisms of soft-endocarp hawthorn.4. The full-length One NAC transcription factor (CpNAC6) and one MYB transcription factor(CpMYB46) are isolated from hawtnorn by PCR. The full-length sequence of CpNAC6has1203bp, encodes400amino acids, the identity of CDS between CpNAC6gene of hawthorn and MdNAC6gene of apple is98.75%. The full-length sequence of CpMYB46has1044bp, encodes347amino acids, the identity of CDS between CpMYB46gene of hawthorn and MdMYB20gene of apple is97.71%.5. Using Nde I and Kpn I to double-digest pRI101-AN DNA vector, link with full-length sequence of GFP gene, construct the plant expression vector pRI101-GFP, including Green fluorescent protein. The full-length sequence of CpNAC6gene and CpMYB46gene are integrated into the multiple cloning sites of the plant expression vector pRI101-GFP to build overexpression vector pOX-CpNAC6and pOX-CpMYB46. The leaf of’Gala’and Nicotianatabacum in vitro plants are transformed by Agrobacterium-mediated leaf disc transformation method. Now, we have one regenerating columnar apple plantlets, twenty regenerating tobacco plantlets for CpNAC6gene, and two regenerating columnar apple plantlets and ninth regenerating tobacco plantlets for CpMYB46gene, which of all have Kanamycin resistance.