| This study，in which Brassica works as plant material, aims to macroscopicallydemonstrate the function of the strip of encoding gene BcMYBogu, transferred intothe Brassica genome after being inserted into plant expression vector as an antisensestrip, plays an important role in affecting the biosynthesis of flavonoid in transgeneicChinese cabbages, of which the flavonoid property was detected, so as to prove theaccommodating function of BcMYBogu transcription factor during the biosynthesispath way of flavonoid.Previous researches mainly focused on the production, the insect resistance and soforth, but few study about the biosynthesis of flavonoid. With the completeArabidopsis thaliana genome sequence published, correlational studies on Brassicawhich shared the same family tree with Arabidopsis thaliana began more based onevidence, providing more theoretical reference for intensive study on physiology andbiochemistry metabolism on it.Chinese cabbage, wildly planted in china, is one of the main dietable vegetablesin people’s daily life. In recent years, as the flavonoid had been recognized as afunctional property preventing all various illnesses and had become one hotspot in thestudy filed, the molecular mechanism of flavonoid was received great attention,something referring to the important function of transcription factors worked the mostprominent effect. In this investigation, a DNA sequence of transcription factor whichmay probably affect the biosynthesis of flavonoid by the transgenic technology wastransferred into the Brassica. And gene transferred plants were cultivated. The wildtypes and blank plasmid transferred plants worked as the blank reference and positivereference respectively. Observed the characters of differently treated samplescarefully and determined the content of extracted flavonoid property, which alsoworked as the proof of the transcription function, providing the basic theoryaccordance and practical basement for getting Chinese cabbages with high productionof flavonoid.The results of study mainly referred in this paper as follows: 1The construction of BcMYBogu-35s-PBI121plant expressing vectorA strand of gene sequence of BcMYBogu, received by digesting theBcMYBogu-pGEM-T plasmid, was connected to the PBI121vector and transferredinto the Escherichia coli, the positive monoclonal bacterium of which was screenedby PCR test, then double-digestion and sequence determination were processedsequentially to the verification of positive bacterium. A BcMYBogu-35s-PBI121plantexpressing vector, reconstructed successfully at last, was effectively transferred intothe Agrobacterium tumefaciens by freezing fusion method, which was simplydemonstrated by PCR test.2The optimization of the transgenic system in Chinese cabbagesIn order to enhance the efficiency of the transgenic system in Brassica, this testworked on these aspects, such as the Kan concentration, the Amp concentration andpH values so on, to optimize the existing transgenic system. The results revealed thatthe Kan concentration played a negative correlation with transferring efficiency, but apositive correlation with the positive percentage. The reasonable concentration rangewas4.0～7.0mg/mL, during which a most suitable Kan concentration—5.0mg/mLwas found. However, the transfering efficiency is still low. During the procession ofdifferentiation, a high Amp concentration prohibited the differentiation of transgenicbud; however, a low Amp concentration played the opposite effect, but could noteffectively curl the growth of Agrobacterium tumefaciens and other kinds ofbacterium. So the Amp concentration must be accommodated according to thetemperature. All in all, when the temperature below25℃, the Amp concentrationshould be set at the range of230～250mg/mL, where it could play the role greatlyinhibited the cultivation of bacterium; Nevertheless, while the temperature above25℃, the most reasonable Amp concentration was270～300mg/mL. All of theseprovided a basis for the tissue cultivation in summer and winter. Besides, the ideal pHvalue range was found at5.6～5.8, neither high nor low is good for burgeoning,which demonstrated by the gradational pH experiment of culture medium.3Identification of transgenic Chinese cabbages， observation of phenotypic character and determination of flavonoid contentThe gene transgenic cabbages were deviated from the fresh lamina at the tissueculture stage, DNA was extracted by CTAB method, the concentration and quality ofwhich were confirmed by the agar gel electrophoresis. Then, a PCR amplify reactionunderwent by the model of DNA to ensure the BcMYBogu sequence had beeninserted into the Brassica genome. The results revealed that the positive efficiency ofthe blank reference—PBI121transferred plant was30%, whileBcMYBogu-35s-PBI121transferred plant is35.7%.Extraction of the flavonoid property of the cabbage lamina could be used for afurther validation, by which the results revealed that the flavonoid content ofBcMYBogu-35s-PBI121transferred plant was lower than any other plants withdifferent treatments, the discrepancy of which is prominent. But the discrepancybetween the blank reference and wild type could not be detected.4The analysis of BcMYBogu gene functionDuring the late period, the exoteric character observation and the detection offlavonoid property in transgenic plant synthetically revealed the up regulation ofBcMYBogu. That means the BcMYBogu-35s-PBI121antisense vector has played aninhibiting role in the translation and expression of BcMYBogu, the function of whichwas partially disabled by BcMYBogu transcription factor. As a result, the ability offlavonoid synthesis in plant was degraded.The investigation was suspended at this stage, owing to the limitation of time andthe research outlay. The late study could be more meticulously adoptingmulti-promoters to start up the expression of gene in plant, by which the curbingefficiency of BcMYBogu-35s-PBI121antisense vector could be enhanced greatly.The certain copy quantity of BcMYBogu and the definition of the inserting point inBrassica genome could be in favor of understanding the function of flavonoidbiosynthesis in plant. |
PDF Full Text Request